CN110636858A

CN110636858A - Human anti-semaphorin 4D antibody

Info

Publication number: CN110636858A
Application number: CN201880029632.7A
Authority: CN
Inventors: E·S·史密斯; A·康尼尔森; M·G·M·斯科瑞文斯; M·帕里斯; M·造德尔
Original assignee: Vassinis Co
Current assignee: Vassinis Co
Priority date: 2017-05-05
Filing date: 2018-05-04
Publication date: 2019-12-31
Anticipated expiration: 2038-05-04
Also published as: MX2019013110A; RU2019131732A; EP3618865A1; CA3061963A1; AU2024204752A1; JP2020518248A; US12006365B2; US20220356249A1; IL270244A; AU2018261947A1; SG11201909466RA; JP2023075271A; IL270244B1; US11427634B2; EP3618865A4; KR20200004324A; US20210032329A1; MX2023003519A; JP7246320B2; JP7460819B2

Abstract

Compositions and methods are provided for treating diseases associated with semaphorin-4D (SEMA4D) pathology, including autoimmune diseases, inflammatory diseases, cancer, neuroinflammatory disorders, and neurodegenerative diseases.

Description

Human anti-semaphorin 4D antibody

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application serial No. 62/501,981, filed on 5/2017, which is incorporated herein by reference in its entirety.

Background

Semaphorin 4D (Semaphorin 4D, SEMA4D), also known as CD100, is a transmembrane protein belonging to the Semaphorin gene family. SEMA4D is expressed as a homodimer on the cell surface, but after cell activation SEMA4D can be released from the cell surface by proteolytic cleavage to produce the soluble form of the protein, sisma 4D, which also has biological activity as SEMA 4D. See Suzuki et al, Nature Rev. Immunol.3:159-167 (2003); kikutani et al, Nature I.M.UNOL.9: 17-23 (2008).

SEMA4D is expressed at high levels in lymphoid organs including the spleen, thymus and lymph nodes, as well as in non-lymphoid organs such as the brain and kidneys. In lymphoid organs, SEMA4D was expressed abundantly on resting T cells, but only weakly on resting B cells and Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs). However, its expression in these cells is up-regulated after activation by various immune stimuli. The release of soluble SEMA4D from immune cells was also increased due to cell activation.

SEMA4D has been implicated in the development of certain cancers (Ch' ng et al, Cancer110: 164-72 (2007); Campos et al, Oncology Letters 5:1527-35 (2013); Kato et al, Cancer sci.102:2029-37(2011)), and several reports suggest that one mechanism of this effect is SEMA4D has an effect in promoting tumor Angiogenesis (Conrotto et al, Blood 105:4321-4329(2005). Basile et al, J biol. chem.282: 34888-. Tumor growth and metastasis involve complex processes of cross-talk between tumor cells, stroma, and immune infiltrates, as well as endothelial cells and vascular structures. SEMA4D is overexpressed in a wide range of tumor types and is also produced by inflammatory cells recruited into the tumor microenvironment. Recent studies have shown that SEMA4D plays a role in the migration, survival, differentiation and organization of different cell types that make up the tumor stroma (Evans et al, Cancer Immunol. Res.3: 689-701 (2015)).

SEMA4D is implicated in neurodegenerative disorders, autoimmune diseases, demyelinating diseases and cancer. In the Central Nervous System (CNS), SEMA4D is expressed on, for example, infiltrating immune cells and oligodendrocyte precursor cells, and its receptor is expressed on, for example, neurons, oligodendrocytes, astrocytes and endothelial cells (Okuno, T. et al, J.Immunol.184: 1499-1506 (2010)). SEMA4D may act as a axonal targeting molecule (Swiercz et al, Neuron 35:51-63(2002)) and may mediate γ -aminobutyric and glutamatergic synaptic development (Paradis et al, Neuron 53:217-232(2007)) as well as have other activities.

SEMA4D has also been shown to play a role in migration and differentiation of neuronal and oligodendrocyte precursor cells, CNS inflammation and neurodegeneration. For example, SEMA4D deficient mice are resistant to the development of Experimental Autoimmune Encephalomyelitis (EAE) (Kumanogoh A et al, Immunity 13: 621-1506 (2000)), and blocking SEMA4D in the case of EAE inhibits microglial activation and neuroinflammation (Okuno, T. et al, J.Immunol.184: 1499-1506 (2010)). Similarly, stimulation of endothelial cells by SEMA4D resulted in the production of the proinflammatory cytokine IL-8(Yang, YH et al, PLoS One 6: e25826 (2011)).

Recent publications have demonstrated that anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments thereof) have efficacy in treating both cancer and neuroinflammatory/neurodegenerative diseases. For example, recent phase I clinical trials with anti-SEMA 4D antibody VX15/2503 demonstrated safety and efficacy in treating solid tumors. See, e.g., Patnaik, A. et AL, Clin. cancer Res.22:827-836(2016) and Southwell AL et AL, neurobiol. Dis 76:46-56 (2015). However, there is still a need for additional anti-SEMA 4D antibodies with improved and/or improved profiles, e.g. with respect to binding characteristics, immunogenicity and/or potency.

Disclosure of Invention

The present disclosure provides compositions and methods for treating diseases associated with semaphorin-4D (SEMA4D) pathology, such as autoimmune diseases, inflammatory diseases, cancer, neuroinflammatory diseases, and neurodegenerative disorders. According to various aspects of the disclosure illustrated herein, there is provided an antibody or antigen-binding fragment thereof that specifically binds to SEMA 4D. According to other aspects of the disclosure illustrated herein, there is provided a method for treating a subject having an autoimmune disease, an inflammatory disease, a cancer, a neuroinflammatory disease, and a neurodegenerative disorder, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment that specifically binds to SEMA4D and neutralizes SEMA4D activity.

In certain aspects, an antibody or antigen-binding fragment thereof that specifically binds to SEMA4D comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, the HCDRs comprising amino acid sequences identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, or identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, except for one, two, three or four amino acid substitutions in one, two or all three of the HCDRs; and wherein the VL comprises complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, the LCDRs comprising amino acid sequences identical to SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively, or identical to SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively, except for one, two, three, or four amino acid substitutions in one, two, or all three of the LCDRs.

In certain aspects, the anti-SEMA 4D antibody or antigen-binding fragment thereof comprises VH complementarity determining regions HCDR1, HCDR2 and HCDR3, said VH complementarity determining regions comprising amino acid sequences identical to SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively; and VL complementarity determining regions LCDR1, LCDR2, and LCDR3, which VL complementarity determining regions comprise amino acid sequences identical to SEQ ID nos. 6,7, and 8, respectively.

In certain aspects, an anti-SEMA 4D antibody or antigen-binding fragment thereof comprises a VH comprising framework regions (HFW) HFW1, HFW2, HFW3, and HFW 4; and a VL comprising framework regions (LFW) LFW1, LFW2, LFW3, and LFW 4. In certain aspects, the framework regions can be derived from human or non-human antibodies.

In certain aspects, the anti-SEMA 4D antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence SEQ ID NO: 1. In certain aspects, an anti-SEMA 4D antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of seq id No. 5. In certain aspects, the anti-SEMA 4D antibody or antigen-binding fragment thereof comprises a VH and a VL comprising the amino acid sequences SEQ ID No. 1 and SEQ ID No. 5, respectively.

In certain aspects, the VH of an anti-SEMA 4D antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 1. In certain aspects, the VL of an anti-SEMA 4D antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 5. In certain aspects, a VH may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 1, and a VL may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 5.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein comprises a heavy chain constant region or fragment thereof fused to the C-terminus of a VH. In certain aspects, the heavy chain constant region or fragment thereof is a human heavy chain constant region. In certain aspects, the heavy chain constant region or fragment thereof can be a human IgG4 constant region. In certain aspects, the heavy chain constant region or fragment thereof is a non-human constant region. In certain aspects, the heavy chain constant region or fragment thereof is a murine IgG1 constant region. In certain aspects, an anti-SEMA 4D antibody or fragment thereof comprises a VH and a VL, wherein a light chain constant region or fragment thereof is fused to the C-terminus of the VL. In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein comprises a light chain constant region or fragment thereof fused to the C-terminus of VL. In certain aspects, the light chain constant region is a human light chain constant region, e.g., a human λ or human κ light chain constant region. In certain aspects, the light chain constant region is a non-human light chain constant region, e.g., a murine λ light chain constant region.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein can be a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, an Fd fragment, a single chain Fv fragment (scFv), or a disulfide-linked Fv fragment (sdFv). In certain aspects, the anti-SEMA 4D antibody or fragment thereof may be multispecific, e.g., bispecific.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein can specifically bind to human SEMA4D, mouse SEMA4D, rat SEMA4D, and/or non-human primate SEMA4D, e.g., cynomolgus SEMA4D and/or marmoset SEMA 4D. In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein may be characterized by a dissociation constant KD of no more than 500nM, 100nM, 50.0nM, 40.0nM, 30.0nM, 20.0nM, 10.0nM, 9.0nM, 8.0nM, 7.0nM, 6.0nM, 5.0nM, 4.0nM, 3.0nM, 2.0nM, 1.0nM, 0.50nM, 0.10nM, 0.050nM, 0.01nM, 0.005nM or 0.001nM that binds to SEMA4D, e.g. human SEMA4D, mouse SEMA4D, rat SEMA4D and/or non-human primate SEMA4D, e.g. cynomolgus monkey SEMA4 monkey 4D and/or marmoset SEMA 4D.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided herein can inhibit SEMA4D from binding to a SEMA4D receptor, e.g., Plexin-B1 (Plexin-B1), Plexin-B2, CD72, or any combination thereof.

In certain aspects, the anti-SEMA 4D antibody or fragment thereof elicits a minimal or no anti-antibody immune response upon administration to a subject.

In certain aspects, the anti-SEMA 4D antibody or fragment thereof comprises a heterologous moiety fused or conjugated thereto. In certain aspects, the heterologous moiety can be, for example, a polypeptide, a cytotoxic agent, a therapeutic agent, a prodrug, a lipid, a carbohydrate, a nucleic acid, a detectable label, a polymer, or any combination thereof. In certain aspects, the heterologous moiety can comprise a binding molecule, an enzyme, a cytokine, a lymphokine, a hormone peptide, or any combination thereof. In another aspect, the heterologous moiety can comprise a radionuclide, a biological toxin, an enzymatically active toxin, or any combination thereof. In another aspect, the heterologous moiety can comprise an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or any combination thereof.

In certain aspects, the present disclosure provides a pharmaceutical composition comprising an anti-SEMA 4D antibody or fragment thereof provided herein, and a carrier.

In certain aspects, the present disclosure provides an isolated polynucleotide or combination of polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA 4D antibody or fragment thereof. In another aspect, the polynucleotide or combination of polynucleotides comprises a nucleic acid sequence encoding the VH of an anti-SEMA 4D antibody or fragment thereof. In another aspect, the polynucleotide or combination of polynucleotides comprises a nucleic acid sequence encoding the VL of an anti-SEMA 4D antibody or fragment thereof. In another aspect, the polynucleotide or combination of polynucleotides comprises nucleic acid sequences encoding the VH and VL of an anti-SEMA 4D antibody or fragment thereof. In another aspect, the present disclosure provides a vector comprising a polynucleotide comprising one or more nucleic acid sequences encoding an anti-SEMA 4D antibody or fragment thereof. In another aspect, the present disclosure provides a host cell comprising the vector. In another aspect, the disclosure provides a method of producing an anti-SEMA 4D antibody or fragment thereof.

In certain aspects of the provided methods, the anti-SEMA 4D antibody or antigen-binding fragment neutralizes SEMA4D in a human subject.

According to various aspects illustrated herein, the present disclosure provides a method of treating a subject having an autoimmune disease or disorder, an inflammatory disease or disorder, a cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment that specifically binds to SEMA4D and neutralizes SEMA4D activity. In certain aspects, the neuroinflammatory disease or disorder is multiple sclerosis. In certain aspects, the neurodegenerative disease or disorder is stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, down syndrome, ataxia, Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), HIV-associated cognitive impairment, CNS lupus, mild cognitive impairment, or a combination thereof. In certain aspects, the autoimmune disease or inflammatory disease is arthritis. In certain aspects, the autoimmune disease or inflammatory disease is rheumatoid arthritis.

Drawings

Figure 1A compares the binding of MAbD2517 and MAbVX15/2503 (both formulated in PBS) to human SEMA4D measured by ELISA.

Figure 1B compares the binding of MAbD2517 and MAbVX15/2503 (both formulated in PBS) to cynomolgus monkey SEMA4D measured by ELISA.

Figure 1C compares the binding of MAbD2517 and MAbVX15/2503 (both formulated in PBS) to marmoset SEMA4D measured by ELISA.

Figure 1D compares the binding of MAbD2517 and MAbVX15/2503 (both formulated in PBS) to mouse SEMA4D measured by ELISA.

Figure 2A shows the ability of MAbD2517 to block binding of human SEMA4D to 293PLXNB1 cells compared to MAbVX 15/2503.

Figure 2B shows the ability of MAbD2517 to block binding of cynomolgus monkey SEMA4D to 293PLXNB1 cells compared to MAbVX 15/2503.

Figure 2C shows the ability of MAbD2517 to block binding of mouse SEMA4D to 293PLXNB1 cells compared to MAbVX 15/2503.

Figure 2D shows the ability of MAbD2517 to block binding of rat SEMA4D to 293PLXNB1 cells compared to MAbVX 15/2503.

FIG. 3A shows the measurement of tumor volume in Balb/c mice treated with control mouse IgG1/2B8 or chimeric antibody MAbD2585(10mg/kg, intraperitoneal, once weekly for 5 weeks).

FIG. 3B shows the survival time of Balb/c mice treated with control mouse IgG1/2B8 or the chimeric antibody MAbD 2585.

FIG. 3C shows tumor regression frequency in Balb/C mice treated with control mouse IgG1/2B8 or chimeric antibody MAbD 2585.

Detailed Description

Definition of

It should be noted that the term "an" entity refers to one or more of that entity; for example "a binding molecule(s)" is understood to represent one or more binding molecule(s). Thus, the terms "a", "an" or "a" and "at least one" are used interchangeably herein.

Further, as used herein, "and/or" should be considered to expressly disclose each of the two specified features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone), and "B" (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, circumcise Dictionary of Biomedicine and molecular Biology, Juo, Pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of cellular Molecular Biology, 3 rd edition, 1999, Academic Press; and Oxford Dictionary of biochemistry And Molecular Biology, revision 2000, Oxford University Press provides the skilled artisan with a comprehensive Dictionary of many of the terms used in this disclosure.

Units, prefixes, and symbols are expressed in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully explained by reference to the specification as a whole.

Whenever an embodiment is described in the language "comprising," further similar embodiments described in "consisting of … …" and/or "consisting essentially of … …" are also provided.

Amino acids are referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB biochemical nomenclature commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition of a mammal in which a population of cells is characterized by unregulated cell growth. Cancers may be classified, for example, as solid or malignant tumors, or hematologic or malignant tumors. Both types can migrate to distant sites in the form of metastatic cancer. Solid tumors can be classified, for example, as sarcomas, carcinomas, melanomas or metastases thereof.

As used herein, "tumor" and "neoplasm" refer to any benign (non-cancerous) or malignant (cancerous) tissue mass resulting from excessive cell growth or proliferation, including precancerous lesions. In certain embodiments, the tumors described herein express SEMA4D receptors, such as plexin-B1, plexin-B2 and/or CD72, and/or may express SEMA 4D.

As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a product of a particular length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products having post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modifications by non-naturally occurring amino acids. The polypeptide may be derived from a biological source, or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

A polypeptide as disclosed herein can have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have that structure. Polypeptides with a defined three-dimensional structure are said to be folded and do not have a defined three-dimensional structure but can adopt many different conformations and are said to be unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is linked to the protein through an oxygen-or nitrogen-containing side chain of an amino acid, such as serine or asparagine.

By "isolated" polypeptide or fragment, variant or derivative thereof, it is meant a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide may be removed from its natural or native environment. As disclosed herein, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique.

As used herein, the term "non-naturally occurring polypeptide" or any grammatical variation thereof is specifically excluded but not limited toOnly byConditional definitions of those forms of the polypeptide that are or may be determined or interpreted by a judge or administrative or judicial authority as "naturally occurring" are excluded.

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the previous polypeptides and any combination thereof. The terms "fragment," "variant," "derivative," and "analog" as disclosed herein include any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide, such as specific binding to an antigen. In addition to specific antibody fragments discussed elsewhere herein, fragments of polypeptides also include, for example, proteolytic fragments as well as deletion fragments. For example, variants of the polypeptides include fragments as described above, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain aspects, a variant may be non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. The variant polypeptide may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives are polypeptides that have been altered so as to exhibit additional characteristics not seen with respect to the original polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a polypeptide can also refer to the subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides containing one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxy lysine can be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine can be substituted for serine; and ornithine may be substituted for lysine.

A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the disclosure do not eliminate binding of the polypeptide or antibody comprising the amino acid sequence to the antigen to which the binding molecule binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al, biochem.32: 1180-1187 (1993); Kobayashi et al, Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

The term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger rna (mrna), cDNA, or plasmid dna (pdna). Polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more segments of nucleic acid, such as DNA or RNA fragments, present in a polynucleotide.

By "isolated" nucleic acid or polynucleotide, it is meant any form of the nucleic acid or polynucleotide that is isolated from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a gel-purified polynucleotide or vector would be considered "isolated". In addition, polynucleotide segments that have been engineered to have cloning restriction sites, such as PCR products, are considered "isolated". Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell, or purified (partially or substantially) polynucleotides in a non-natural solution such as buffer or saline. An isolated RNA molecule includes in vivo or in vitro RNA polynucleotide transcripts, where the transcripts are not those that would be found in nature. Isolated polynucleotides or nucleic acids also include synthetically produced said molecules. In addition, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

As used herein, the term "non-naturally occurring polynucleotide" or any grammatical variation thereof is specifically excluded but not limited toOnly byConditional definitions of those forms of the nucleic acid or polynucleotide that are or may be determined or interpreted by a judge or administrative or judicial authority as "naturally occurring" are excluded.

As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it may be considered part of the coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a single vector may encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region separately. In addition, the vector, polynucleotide or nucleic acid may include a heterologous coding region, fused or unfused to another coding region. Heterologous coding regions include, without limitation, those that encode specialized elements or motifs such as secretion signal peptides or heterologous functional domains.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may typically include a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. The operable association is when: the coding region of a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in a manner that places expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or with the ability of the DNA template to be transcribed. Thus, if a promoter is capable of effecting transcription of a nucleic acid encoding a polypeptide, the promoter region will be operably associated with that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may also be operably associated with the polynucleotide to direct cell-specific transcription.

Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, in combination with intron-a), simian virus 40 (early promoter), and retroviruses, such as Rous sarcoma virus (Rous sarcoma). Other transcriptional control regions include those derived from vertebrate genes, such as actin (actin), heat shock proteins, bovine growth hormone, and rabbit β -globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

In other embodiments, the polynucleotide may be RNA, e.g., in the form of messenger RNA (mrna), transfer RNA, or ribosomal RNA.

The polynucleotide and nucleic acid coding regions may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. One of ordinary skill in the art will appreciate that a polypeptide secreted by a vertebrate cell can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the full or "full-length" polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of that sequence that retains the ability to direct secretion of the polypeptide with which it is operably associated, is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless a full-size antibody is specifically mentioned, the term "binding molecule" encompasses a full-size antibody as well as antigen-binding subunits, fragments, variants, analogs or derivatives of the antibody, e.g., engineered antibody molecules or fragments that bind antigen in a similar manner as antibody molecules, but using a different backbone.

As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a receptor, such as an epitope or antigenic determinant. As further described herein, a binding molecule can comprise one or more "antigen binding domains" as described herein. A non-limiting example of a binding molecule is an antibody or fragment thereof that retains antigen-specific binding.

As used herein, the term "binding domain" or "antigen binding domain" refers to a region of a binding molecule that is necessary for, and sufficient to specifically bind an epitope. For example, an "Fv", such as a variable heavy chain and a variable light chain of an antibody, in the form of two separate polypeptide subunits or in single chain form, is considered a "binding domain". Other binding domains include, but are not limited to, the variable heavy chain (VHH) of antibodies derived from camelid species, or the six immunoglobulin Complementarity Determining Regions (CDRs) expressed in the fibronectin backbone. A "binding molecule" as described herein may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more "antigen binding domains".

The terms "antibody" and "immunoglobulin" may be used interchangeably herein. The antibody (or fragment, variant or derivative thereof as disclosed herein) comprises at least a heavy chain variable region (for camelid species) or at least a heavy chain variable region and a light chain variable region. The basic immunoglobulin structure in vertebrate systems is relatively well understood. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988). Unless stated otherwise, the term "antibody" encompasses anything ranging from a small antigen-binding fragment of an antibody to a full-size antibody, such as an IgG antibody comprising two complete heavy chains and two complete light chains, an IgA antibody comprising four complete heavy chains and four complete light chains, and optionally comprising a J chain and/or secretory component, or an IgM antibody comprising ten or twelve complete heavy chains and ten or twelve complete light chains, and optionally comprising a J chain.

As will be discussed in more detail below, the term "immunoglobulin" includes a wide variety of classes of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclass (e.g., gamma 1-gamma 4 or alpha 1-alpha 2) among them. The nature of this chain identifies the "class" of antibodies as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulins (isotypes) such as IgG₁、IgG₂、IgG₃、IgG₄、IgA₁、IgA₂Etc. are well characterized and are known to confer functional specialization. Modified forms of each of these classes and isoforms can be readily identified by the skilled artisan in view of the present disclosure, and are therefore within the scope of the present disclosure.

Light chains are classified as either kappa or lambda (kappa, lambda). Each heavy chain class can be associated with a kappa or lambda light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonding or non-covalent bonding. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, such as IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a "Y" structure, also referred to herein as the "H2L 2" structure.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in terms of function. In this regard, it is understood that the variable regions of both the Variable Light (VL) chain portion and the Variable Heavy (VH) chain portion determine antigen recognition and specificity. In contrast, the constant region of the light Chain (CL) and the constant region of the heavy chain (CH1, CH2, or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

As indicated above, the variable regions (i.e., "binding domains") allow the binding molecules to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH regions or subsets of Complementarity Determining Regions (CDRs) of a binding molecule, such as an antibody, combine to form variable regions that define a three-dimensional antigen binding site. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains. Some antibodies form larger structures. For example, IgA can form molecules that include: two H2L2 units, a J chain and a secretory component, all covalently linked by a disulfide bond; and IgM can form a pentameric or hexameric molecule comprising: five or six H2L2 units, and optionally J chains, are covalently linked by disulfide bonds.

The six "complementarity determining regions" or "CDRs" present in an antibody antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the binding domain when the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining amino acids in the binding domain, referred to as the "framework" region, exhibit little intermolecular variability. The framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β -sheet structure. Thus, the framework regions serve to form a framework that provides for positioning the CDRs in the correct orientation through inter-chain non-covalent interactions. The binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and framework regions, respectively, of any given heavy or light chain variable region can be readily identified by one of ordinary skill in the art, as they have been defined in a variety of different ways (see "Sequences of Proteins of Immunological Interest," Kabat, E. et al, U.S. department of Health and Human Services, (1983); and Chothia and Lesk, J.mol.biol.,196:901-917(1987), which are incorporated herein by reference in their entirety).

In the case where there are two or more definitions for terms used and/or accepted in the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al, J.mol.biol.196:901-917(1987), which are incorporated herein by reference. The Kabat definition and the Chothia definition include overlapping amino acids or amino acid subgroups when compared to each other. However, application of any definition (or other definition known to those of ordinary skill in the art) to a CDR that refers to an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. Suitable amino acids encompassing the CDRs as defined by each of the above-cited references are set forth below in table 1 as a comparison. The exact number of amino acids that encompass a particular CDR will vary depending on the sequence and size of the CDR. In view of the variable region amino acid sequence of an antibody, one skilled in the art can determine which amino acids comprise a particular CDR in a routine manner.

Table 1: CDR definition¹

	Kabat	Chothia
			VH CDR1	31-35	26-32
VH CDR2	50-65	52-58
			VH CDR3	95-102	95-102
VL CDR1	24-34	26-32
			VL CDR2	50-56	50-52
VL CDR3	89-97	91-96

¹The numbering of all CDR definitions in Table 1 is according to the numbering convention set forth by Kabat et al (see below).

Immunoglobulin variable regions may also be represented using the IMGT information system (www:// IMGTV-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X, et al, Nucl. acids sRs.36: W503-508 (2008).

Kabat et al also define a numbering system for the variable region sequences that can be applied to any antibody. One of ordinary skill in the art can explicitly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and human Services, "Sequence of Proteins of Immunological Interest" (1983). However, unless the Kabat numbering system is explicitly indicated, consecutive numbering is used for all amino acid sequences in the present disclosure.

Binding molecules (e.g., antibodies or antigen binding fragments, variants or derivatives thereof) include, but are not limited to, polyclonal, monoclonal, human, humanized or chimeric antibodies, single chain antibodies, epitope binding fragments, such as Fab, Fab 'and F (ab')₂Fd, Fv, single chain Fv (scFv), single chain antibody, disulfide linked Fv (sdFv), fragments comprising a VL region or a VH region, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. patent No. 5,892,019. The immunoglobulin or antibody molecules encompassed by the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

By "specifically binds," it is generally meant that a binding molecule, such as an antibody or fragment, variant or derivative thereof, binds an epitope through its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when: it binds to a random unrelated epitope more readily than it would bind to that epitope through its antigen binding domain. The term "specificity" is used herein to characterize the relative affinity with which a binding molecule binds to an epitope. For example, a binding molecule "a" may be considered to have a higher specificity for a given epitope than a binding molecule "B", or may be said to bind epitope "C" with a higher specificity than it has for a related epitope "D".

A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, disclosed herein can be referred to as being less than or equal to 5X10^-2 sec^-1、10^-2sec^-1、5X10^-3 sec^-1、10^-3sec^-1、5X10^-4 sec^-1、10^-4sec^-1、5X10^-5 sec^-1Or 10^-5sec^-1、5X10^-6 sec^-1、10^-6sec^-1、5X10^-7 sec^-1Or 10^-7sec^-1The off rate (k (off)) binds to the target antigen.

A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be referred to as being greater than or equal to 10³M^-1sec^-1、5X10³ M^-1sec^-1、10⁴M^-1sec^-1、5X10⁴ M^-1sec^-1、10⁵M^-1sec^-1、5X10⁵ M^-1sec^-1、10⁶M^-1sec^-1Or 5X10⁶ M^-1sec^-1Or 10⁷M^-1sec^-1The association rate (k (on)) of (a) binds to the target antigen.

A binding molecule such as an antibody or fragment, variant or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if: to the extent that it blocks the binding of the reference antibody or antigen-binding fragment to the epitope to some extent, it preferentially binds that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. A binding molecule can be said to inhibit binding of a reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% in a competitive manner.

As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope to, for example, one or more binding domains of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988), at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of binding domains and an antigen. See, e.g., Harlow, at pages 29-34. Avidity is related to the affinity of individual binding domains in a population for a particular epitope, and also to the valency of the immunoglobulin and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, will be a high avidity interaction. The interaction between bivalent monoclonal antibodies and receptors present at high density on the cell surface will also have high affinity.

The binding molecules as disclosed herein or antigen binding fragments, variants or derivatives thereof may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, such as an antibody or fragment, variant or derivative thereof, having specificity for one antigen to react with a second antigen; which is a measure of the correlation between two different antigenic substances. Thus, a binding molecule is cross-reactive if it binds to an epitope other than the epitope that induced its formation. Cross-reactive epitopes and inducible epitopes often contain many of the same complementary structural features and, in some cases, can actually fit better than the original epitope.

Binding molecules such as antibodies or fragments, variants or derivatives thereof may also be described or specified in terms of their binding affinity for an antigen. For example, the binding molecule may be no greater than 5x10^-2M、10^-2M、5×10^-3M、10^-3M、5×10^-4M、10^- ⁴M、5×10^-5M、10^-5M、5×10^-6M、10^-6M、5×10^-7M、10^-7M、5×10^-8M、10^-8M、5×10^-9M、10^-9M、5×10^-10M、10^-10M、5×10^-11M、10^-11M、5×10^-12M、10^-12M、5×10^-13M、10^-13M、5×10^-14M、10^-14M、5×10^- ¹⁵M, or 10^-15Dissociation constant of M or K_DBinding to an antigen.

Antibody fragments including single chain antibodies or other binding domains may be present alone or in combination with one or more of the following: a hinge region, a CH1, a CH2, a CH3 or CH4 domain, a J chain, or a secretory component. Also included are antigen binding fragments that may include any combination of the variable regions and one or more of the following: a hinge region, a CH1, a CH2, a CH3 or CH4 domain, a J chain, or a secretory component. Binding molecules such as antibodies or antigen-binding fragments thereof can be from any animal source, including birds and mammals. The antibody may be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, in terms of origin, the variable region may be a cartilaginous fish variable region (e.g., from shark). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library, or antibodies isolated from an animal that is transgenic for one or more human immunoglobulins and in some cases may express an endogenous immunoglobulin, and in some cases does not express an endogenous immunoglobulin, as described below and, for example, in U.S. patent No. 5,939,598 to Kucherlapati et al.

As used herein, the term "heavy chain subunit" includes amino acid sequences derived from an immunoglobulin heavy chain, and a binding molecule, such as an antibody, comprising a heavy chain subunit includes at least one of: a VH region, a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge) domain, a CH2 domain, a CH3 domain, a CH4 domain, or variants or fragments thereof. For example, in addition to VH regions, binding molecules such as antibodies or fragments, variants or derivatives thereof may also include a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain aspects, a binding molecule, such as an antibody or fragment, variant or derivative thereof, may also include a CH3 domain and a CH4 domain in addition to a VH region; or a CH3 domain, a CH4 domain, and a J chain. Furthermore, binding molecules for use in the present disclosure may lack certain constant region portions, such as all or a portion of the CH2 domain. One of ordinary skill in the art will appreciate that these domains (e.g., heavy chain subunits) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule.

The heavy chain subunit of a binding molecule (e.g., an antibody or fragment thereof) can include domains derived from different immunoglobulin molecules. For example, a heavy chain subunit of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain subunit can include a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain subunit can comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term "light chain subunit" includes amino acid sequences derived from immunoglobulin light chains. The light chain subunit includes at least one of: VL or CL (e.g., C κ or C λ) domains.

Binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) may be described or specified in terms of the epitope or portion of the antigen to which they recognize or specifically bind. The portion of the target antigen that specifically interacts with the antigen-binding domain of the antibody is an "epitope" or "antigenic determinant. Depending on the size, conformation, and type of antigen, the target antigen may comprise a single epitope or at least two epitopes, and may include many epitopes.

As indicated previously, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH region" includes the amino-terminal variable region of an immunoglobulin heavy chain, and the term "CH 1 domain" includes the first (amino-terminal-most) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH region and is amino terminal to the hinge region of a typical immunoglobulin heavy chain molecule.

As used herein, the term "CH 2 domain" includes that portion of the heavy chain molecule which extends, for example, from about amino acid 244 to amino acid 360 of an IgG antibody (amino acids 244 to 360, the Kabat numbering system; and amino acids 231-. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 amino acids. Certain immunoglobulin classes, such as IgM, also include the CH4 region.

As used herein, the term "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises about 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group which can form a disulfide bond or a disulfide bridge with a second thiol group. In certain IgG molecules, the CH1 region and the CL region are linked by disulfide bonds, and where the Kabat numbering system is used, the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 (positions 226 or 229, EU numbering system).

As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species, while the constant region (which may be intact, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate), while the constant region is a human constant region.

The term "multispecific antibody" or "bispecific antibody" refers to an antibody having binding domains directed to two or more different epitopes within a single antibody molecule. In addition to typical antibody structures, other binding moleculesCan also be constructed to have two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of bispecific antibody secreting cell lines. Bispecific antibodies can also be constructed by recombinant means. (And Heiss, Future Oncol.6:1387-94 (2010); mabry and Snavely, IDrugs.13:543-9 (2010)). Bispecific antibodies may also be minibifunctional antibodies.

As used herein, the term "engineered antibody" refers to an antibody in which the variable regions in the heavy and light chains, or both, are altered by at least partial substitution of one or more amino acids in the CDRs or framework regions. In certain aspects, the entire CDR from an antibody of known specificity can be grafted into the framework region of a heterologous antibody. Although the alternating CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, the CDRs may also be derived from a different class of antibody, e.g. from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted into a human heavy chain framework region or a light chain framework region is referred to herein as a "humanized antibody". In certain aspects, not all CDRs are replaced with complete CDRs from the donor variable region, yet the antigen binding capacity of the donor can still be transferred into the recipient variable region. In view of the explanations set forth in, for example, U.S. Pat. nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it would be well within the ability of those skilled in the art to obtain functional engineered or humanized antibodies by performing routine experimentation or by trial and error testing.

As used herein, the term "engineering" includes manipulation of a nucleic acid or polypeptide molecule by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or some combination of these techniques).

As used herein, the terms "connected," "fused," or other grammatical equivalents may be used interchangeably. These terms refer to the bringing together of two or more elements or components by whatever means including chemical conjugation or recombinant means. By "in-frame fusion" is meant joining two or more polynucleotide Open Reading Frames (ORFs) to form a continuous longer ORF in a manner that maintains the translational reading frame of the original ORF. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (which segments are not normally so joined in nature). Although the reading frame is thus continuous throughout the fused segment, the segments may be physically or spatially separated by, for example, in-frame linker sequences. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused in-frame, but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

In the case of polypeptides, a "linear sequence" or "sequence" is a sequence of amino acids in a polypeptide in the amino-terminal to carboxy-terminal direction, wherein the amino acids adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino-terminal" or "N-terminal" to another portion of the polypeptide is that portion that occurs earlier in the contiguous polypeptide chain. Similarly, a portion of a polypeptide that is "carboxy-terminal" or "C-terminal" to another portion of the polypeptide is that portion that occurs later in a contiguous polypeptide chain. For example, in a typical antibody, the variable region is "N-terminal" to the constant region, and the constant region is "C-terminal" to the variable region.

The term "expression" as used herein refers to the process by which a gene produces a biochemical, e.g., a polypeptide. The process includes any manifestation of the functional presence of a gene within a cell, including, without limitation, gene knockdown and both transient and stable expression. This includes, but is not limited to, transcription of genes into messenger rna (mRNA), and translation of the mRNA into polypeptides. If the final desired product is a biochemical, expression includes production of that biochemical and any precursors. Expression of a gene will produce a "gene product". As used herein, a gene product can be a nucleic acid, such as a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications, such as polyadenylation, or polypeptides with post-translational modifications, such as methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

As used herein, the term "anti-SEMA 4D binding molecule" refers to a molecule, such as an antibody or antigen-binding fragment, variant or derivative thereof, that specifically binds to SEMA 4D. Unless explicitly mentioned to a full-size antibody such as a naturally occurring antibody, the term "anti-SEMA 4D antibody" encompasses full-size antibodies as well as antigen-binding fragments and antigen-binding fragments, variants, analogs or derivatives of said antibodies, e.g. naturally occurring antibodies or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a similar manner as antibody molecules.

Terms such as "treating" or "to treat" or "alleviate" or "to alleviate" refer to therapeutic measures that cure, alleviate the symptoms of, and/or halt or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as "preventing/prevention", "avoiding", "deterring", and the like, refer to prophylactic or preventative measures to prevent the manifestation of an undiagnosed targeted pathological condition or disorder. Thus, a "subject in need of treatment" can include a subject already suffering from a disease; patients predisposed to disease; and patients to be prevented. As used herein, phrases such as "a subject who will benefit from therapy" and "an animal in need of treatment" include subjects, such as mammalian subjects, who will benefit from administration of a therapy as described herein.

The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or condition in a subject or mammal.

By "subject" or "individual" or "animal" or "patient" or "mammal", it is meant any subject, particularly a mammalian subject, in need of diagnosis, prognosis or therapy. Mammalian subjects include humans, domestic animals, farm and zoo animals, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

Description of the target polypeptide-SEMA 4D

As used herein, the terms "semaphorin-4D", "SEMA 4D" and "SEMA 4D polypeptide" are used interchangeably, and "SEMA 4D" and "SEMA 4D" are also used interchangeably. In certain embodiments, SEMA4D is expressed on the surface of a cell, or secreted by a cell. In another embodiment, SEMA4D is of the membrane bound type. In another embodiment, SEMA4D is soluble, for example, tsema 4D. In another embodiment, SEMA4D may comprise full-size SEMA4D or a fragment thereof, or a SEMA4D variant polypeptide, wherein a fragment of SEMA4D or a SEMA4D variant polypeptide retains some or all of the functional properties of full-size SEMA 4D.

The full-size human SEMA4D protein is a homodimeric transmembrane protein composed of two 150kDa polypeptide chains. SEMA4D belongs to the cell surface receptor family of semaphorins and is also known as CD 100. Both human SEMA4D/Sema4D and mouse SEMA4D/Sema4D can be proteolytically cleaved from their transmembrane forms to yield the 120kDa soluble form, yielding two Sema4D subtypes (Kumanogoh et al, J.cell Science116:3464 (2003)). Semaphorins consist of soluble and membrane-bound proteins originally identified as axon-homing factors that play an important role in establishing precise connections between neurons and their appropriate targets. Structurally considered to be a class IV semaphorin, SEMA4D consists of an amino-terminal signal sequence followed by a characteristic 'SEMA' domain containing 17 conserved cysteine residues, an Ig-like domain, a lysine-rich segment, a hydrophobic transmembrane domain, and a cytoplasmic tail.

The SEMA4D polypeptide includes a signal sequence of about 13 amino acids followed by a semaphorin domain of about 512 amino acids, an immunoglobulin-like (Ig-like) domain of about 65 amino acids, a lysine-rich segment of 104 amino acids, a hydrophobic transmembrane region of about 19 amino acids, and a cytoplasmic tail of 110 amino acids. The consensus site for tyrosine phosphorylation in the cytoplasmic tail supports the predicted association of SEMA4D with tyrosine kinases (Schlossman et al (1995) Leucocyte Typing V (Oxford University Press, Oxford)).

SEMA4D is known to have at least three functional receptors, namely plexin-B1, plexin-B2 and CD 72. Plexin-B1 is expressed in non-lymphoid tissues and has been shown to be a high affinity (1nM) receptor for SEMA4D (Tamagnone et al, Cell99:71-80 (1999)). It has been shown that stimulation of plexin-B1 signaling by SEMA4D induces growth cone collapse of neurons and induces process extension collapse and apoptosis in oligodendrocytes (Giraudon et al, J.Immunol.172: 1246-. After binding to SEMA4D, plexin-B1 signaling mediates inactivation of R-Ras leading to integrin-mediated reduction of adhesion to the extracellular matrix, and activation of RhoA leading to Cell collapse caused by reorganization of the cytoskeleton (Kruger et al, NatureRev. mol. Cell biol.6:789-800 (2005); Passterkamp, TRENDS in Cell Biology 15:61-64 (2005)). plexin-B2 has a moderate affinity for SEMA4D, and it has been recently reported that plexin-B2 is expressed on keratinocytes and activates SEMA4D positive γ δ T cells to promote epithelial repair (Witherden et al, Immunity 37:314-25 (2012)).

In lymphoid tissues, CD72 was used as a low affinity (300nM) SEMA4D receptor (Kumanogoh et al, Immunity 13:621-631 (2000)). B cells and Antigen Presenting Cells (APC) express CD72, and anti-CD 72 antibodies have many of the same effects as sSEMA4D, such as enhancing CD 40-induced B cell responses and B cell CD23 shedding. CD72 is thought to act as a negative regulator of B cell response by recruiting the tyrosine phosphatase SHP-1, which can associate with many inhibitory receptors. The interaction of SEMA4D with CD72 resulted in the dissociation of SHP-1 and the loss of this negative activation signal. SEMA4D has been shown to promote T cell stimulation and B cell aggregation and survival in vitro. Addition of SEMA4D expressing cells or sSEMA4D enhanced CD 40-induced B cell proliferation and immunoglobulin production in vitro and accelerated antibody responses in vivo (Ishida et al, Inter. Immunol.15: 1027-42 (2003); Kumanogoh and H.Kukukukukukukuutani, Trends in Immunol.22:670-676 (2001)). The enhancement of CD 40-induced DC maturation by sSEMA4D, including upregulation of costimulatory molecules and increased secretion of IL-12. Furthermore, sSEMA4D inhibited immune cell migration, which was reversed by addition of a blocking anti-SEMA 4D mouse antibody (Elhabazi et al, J.Immunol.166:4341-4347 (2001); Delaire et al, J.Immunol.166:4348-4354 (2001)).

Sema4D is expressed at high levels in lymphoid organs including the spleen, thymus and lymph nodes, as well as in non-lymphoid organs such as the brain, heart and kidney. In lymphoid organs Sema4D is expressed abundantly on resting T cells, but only weakly on resting B cells and Antigen Presenting Cells (APCs) such as Dendritic Cells (DCs).

Cell activation increased surface expression of SEMA4D and production of soluble SEMA4D (issema 4D). The expression pattern of SEMA4D suggests that it plays an important physiological as well as pathological role in the immune system. SEMA4D has been shown to promote B cell activation, aggregation and survival; enhancement of CD 40-induced proliferation and antibody production; (ii) enhancing antibody response to T cell-dependent antigens; (ii) increased T cell proliferation; maturation of dendritic cells and enhanced ability to stimulate T cells; and are directly involved in demyelination and axonal degeneration (Shi et al, Imm unit 13:633-642 (2000); Kumanogoh et al, J.Immunol.169:1175-1181 (2002); and Watanabe et al, J.Immunol.167:4321-4328 (2001)).

Human anti-SEMA 4D antibody

The present disclosure provides an anti-SEMA 4D binding molecule, e.g., an anti-SEMA 4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof having a heavy chain variable region (VH) and a light chain variable region (VL) related to or identical to the VH and VL comprising the amino acid sequences SEQ ID NO:1 and SEQ ID NO:5, respectively. In certain aspects, the provided binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) are fully human binding molecules.

In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) provided herein has a VH region and a VL region comprising amino acid sequences at least about 80%, about 85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the VH and VL regions of a reference anti-SEMA 4D antibody molecule having VH and VL regions comprising amino acid sequences SEQ ID No. 1 and SEQ ID No. 5, respectively.

In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) can inhibit the interaction of SEMA4D with its receptor, e.g., plexin-B1, plexin-B2, or CD 72. In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) can inhibit SEMA 4D-mediated plexin-B1 signaling.

In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) competitively inhibits binding of a reference antibody comprising a variable heavy chain region (VH) comprising amino acid sequence SEQ ID NO:1 and a variable light chain region (VL) comprising amino acid sequence SEQ ID NO:5 to SEMA 4D. In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) binds the same SEMA4D epitope as a reference antibody comprising a VH comprising amino acid sequence SEQ ID No. 1 and a VL comprising amino acid sequence SEQ ID No. 5. In certain aspects, the VH of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) comprises three Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises three CDRs LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, except having at least one, two, three, four, five, or six single conservative amino acid substitutions in one or more of the CDRs. In certain aspects, the CDRs comprise the amino acid sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively.

In certain aspects, the VH of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 1, and the VL of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 5. In certain aspects, the VH comprises the amino acid sequence SEQ ID NO. 1 and the VL comprises the amino acid sequence SEQ ID NO. 5.

Also provided herein are polypeptides encoding an anti-SEMA 4D antibody or antigen-binding fragment, variant or derivative thereof as described herein, polynucleotides encoding the polypeptides, vectors comprising the polynucleotides, and host cells comprising the vectors or polynucleotides, e.g., for use in the production of an anti-SEMA 4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof as provided herein.

anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof, suitable biologically active variants) are provided by the present disclosure and can be prepared and used according to the methods provided herein or according to methods well known to those of ordinary skill in the art. The variants retain some of the desired binding properties of the reference anti-SEMA 4D antibody provided herein. Methods for making antibody variants are generally available in the art.

Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, e.g., Walker and Gaastra eds (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); kunkel, Proc.Natl.Acad.Sci.USA 82:488-492 (1985); kunkel et al, methods enzymol.154: 367-; sambrook et al (1989) Molecular Cloning A laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. nos. 4,873,192; and references cited therein; which is incorporated herein by reference. Guidance as to suitable amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff et al (1978), Atlas of Protein Sequence and Structure (Natl. biomed. Res. Foundation, Washington, D.C.), p.345-352, which is incorporated herein by reference in its entirety. The model of Dayhoff et al uses a point-accepting mutation (PAM) amino acid similarity matrix (PAM250 matrix) to determine suitable conservative amino acid substitutions. In certain aspects, conservative substitutions are used, such asOne amino acid is exchanged for another amino acid with similar properties. Examples of conservative amino acid substitutions as taught by the PAM250 matrix of the model of Dayhoff et al include, but are not limited to And

in constructing variants of an anti-SEMA 4D binding molecule, e.g. an antibody or antigen-binding fragment thereof, a polypeptide of interest, modified such that the variants continue to have desired properties, e.g. are capable of specifically binding to, e.g. SEMA4D, e.g. human SEMA4D, non-human primate SEMA4D (e.g. cynomolgus, marmoset and/or rhesus SEMA4D) and/or rodent SEMA4D (e.g. mouse and/or rat SEMA4D) expressed on the surface of or secreted by a cell, wherein the binding molecule, e.g. antibody or fragment, variant or derivative thereof has SEMA4D binding, receptor blocking or neutralising activity as described herein. In certain aspects, mutations made in DNA encoding the variant polypeptide maintain the reading frame and do not create regions of complementarity that can give rise to secondary mRNA structure. See european patent application publication No. 75,444.

Methods for measuring the binding specificity and/or activity of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) include, but are not limited to, standard binding assays, including competitive binding assays, assays for monitoring immunoglobulin secretion by T cells or B cells, T cell proliferation assays, apoptosis assays, ELISA assays, and the like. See, e.g., WO 93/14125; shi et al, Immunity 13:633-642 (2000); kumanogoh et al, J.Immunol.169:1175-1181 (2002); watanabe et al, J.Immunol.167:4321-4328 (2001); wang et al, Blood 97: 3498-; and Giraudon et al, J.Immunol.172:1246-1255(2004), all of which are incorporated herein by reference.

When discussing herein whether any particular polypeptide disclosed herein, including a constant region, CDR, VH region, or VL region, is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to another polypeptide, the% identity can be determined using methods and computer programs/software known in the art. Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm (Smith and Waterman, adv. appl. Math.2: 482-. 489(1981)), by the global alignment algorithm (Needleman and Wunsch, J.mol. biol.48:443(1970)), by the similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444 (1988); Altschul et al, Nucl. acids Res.25: 3389-. 402(1997)), by computerized implementation programs in these algorithms, which usually use default settings (e.g.Wisconsin Genetics software package (Genetics Computer Group,575Science Dr., Madison, Wis.), or by manual alignment and Current (see, for example, Biology, Inc.), by visual inspection (see, for example, Biology, Inc., Biocolour et al, 1994).

For the purposes of this disclosure, percent sequence identity can be determined using a search using the Smith-Waterman homology search algorithm using a gap opening penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. A variant may differ, for example, by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10 amino acid residues, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue, from a reference anti-SEMA 4D antibody (e.g., MAb 2517 provided herein).

In certain aspects, the present disclosure provides an antibody or antigen-binding fragment, variant, or derivative thereof that specifically binds to SEMA4D, wherein the antibody or fragment thereof comprises a VH and a VL. In certain aspects, the VH comprises complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences identical to SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4, respectively, or identical to SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4, respectively, except for one, two, three, four, five, or six amino acid substitutions in one, two, or all three of the HCDRs. In certain aspects, the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences identical to SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively. In certain aspects, the VL comprises complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences identical to SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively, or identical to SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively, except for one, two, three, four, five, or six amino acid substitutions in one, two, or all three of the LCDRs. In certain aspects, LCDR1, LCDR2, and LCDR3 comprise amino acid sequences identical to SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively. In certain aspects, VH further comprises framework regions (HFW) HFW1, HFW2, HFW3, and HFW4, and VL further comprises framework regions (LFW) LFW1, LFW2, LFW3, and LFW 4. In certain aspects, the framework regions are derived from human antibodies. In certain aspects, e.g., when the antibody is to be used in a non-human model system, the framework regions are derived from a non-human antibody, e.g., a mouse antibody. In certain aspects, the VH comprises the amino acid sequence SEQ ID NO 1. In certain aspects, the VL comprises the amino acid sequence SEQ ID NO 5.

In certain aspects, the present disclosure provides an antibody, e.g., a fully human antibody or antigen-binding fragment, variant, or derivative thereof comprising a VH and a VL that specifically binds semaphorin-4D (SEMA 4D). In certain aspects, the VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No. 1. In certain aspects, the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO 5. In certain aspects, the VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No. 1; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 5. In certain aspects, the VH comprises the amino acid sequence SEQ ID NO. 1 and the VL comprises the amino acid sequence SEQ ID NO. 5. In certain aspects, the VH and VL are fully human VH and VL.

In certain aspects, an anti-SEMA 4D antibody or antigen-binding fragment, variant or derivative thereof as provided herein may specifically bind to human SEMA4D, rodent SEMA4D, e.g. mouse SEMA4D and/or rat SEMA4D, and/or non-human primate SEMA4D, e.g. cynomolgus SEMA4D and/or marmoset SEMA 4D.

In certain aspects, the antibody or fragment, variant, or derivative thereof further comprises a heavy chain constant region, or fragment, variant, or derivative thereof, fused to the C-terminus of the VH. The heavy chain constant region or fragment thereof may be derived from any species, but in certain aspects the heavy chain constant region or fragment, variant or derivative thereof is a human heavy chain constant region or is derived from a human heavy chain constant region, for example a human IgG1, IgG2, IgG3, IgG4, IgA, IgE or IgM constant region. In certain aspects, the heavy chain constant region is a human IgG4 constant region or a fragment, variant, or derivative thereof. In certain aspects, for example when the provided anti-SEMA 4D antibody or fragment thereof is to be used in a non-human model system, the heavy chain constant region or fragment thereof may be a non-human heavy chain constant region, for example a murine heavy chain constant region such as a murine IgG1 constant region.

In certain aspects, the antibody, or fragment, variant, or derivative thereof, further comprises a light chain constant region, or fragment, variant, or derivative thereof, fused to the C-terminus of the VL. The light chain constant region or fragment thereof can be derived from any species, but in certain aspects, the light chain constant region or fragment thereof is derived from a human light chain constant region, e.g., a human kappa or lambda constant region. In certain aspects, the light chain constant region, or fragment, variant, or derivative thereof, is a human λ light chain constant region, or is derived from a human λ light chain constant region. In certain aspects, the light chain constant region is a human λ constant region. In certain aspects, for example when the provided anti-SEMA 4D antibody or fragment thereof is to be used in a non-human model system, the light chain constant region or fragment thereof may be a non-human light chain constant region, e.g., a murine light chain constant region such as a murine λ constant region.

In certain aspects, the present disclosure provides an anti-SEMA 4D antibody fragment comprising a VH region and a VL region as described above. In certain aspects, the fragment can be, for example, a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, an Fd fragment, a single chain Fv fragment (scFv), or a disulfide-linked Fv fragment (sdFv).

In certain aspects, an anti-SEMA 4D antibody or fragment, variant, or derivative thereof provided by the present disclosure can be multispecific, e.g., bispecific. In addition to the binding properties of the SEMA4D antibodies as provided herein, multispecific antibodies or fragments thereof as provided herein may also bind to additional SEMA4D epitopes or may bind to other unrelated epitopes.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof as provided herein can be characterized by a dissociation constant K_DA binding affinity of no more than 500nM, 100nM, 50.0nM, 40.0nM, 30.0nM, 20.0nM, 10.0nM, 9.0nM, 8.0nM, 7.0nM, 6.0nM, 5.0nM, 4.0nM, 3.0nM, 2.0nM, 1.0nM, 0.50nM, 0.10nM, 0.050nM, 0.01nM, 0.005nM or 0.001nM specifically binds to SEMA4D such as human SEMA4D, mouse SEMA4D, cynomolgus SEMA4D, marmoset SEMA4D, or any combination thereof. For measuring K of antibody or fragment thereof against SEMA4D_DSuch as BIACORE, are well known to those of ordinary skill in the art.

In certain aspects, an anti-SEMA 4D antibody or fragment thereof provided by the present disclosure can inhibit SEMA4D from binding to a SEMA4D receptor, e.g., plexin-B1, plexin-B2 and/or CD 72.

In certain aspects, the anti-SEMA 4D antibody or antigen-binding fragment, variant, or derivative thereof provided herein is a fully human anti-SEMA 4D antibody or antigen-binding fragment, variant, or derivative thereof, and elicits minimal or no anti-antibody immune response upon administration to a human subject. Methods for measuring an anti-antibody immune response in a subject are well known in the art. See, e.g., Darwish, IA, int.J.biomed.Sci.2:217-235 (2006).

The constant region of the anti-SEMA 4D antibody can be mutated in a number of ways to alter effector function. See, for example, U.S. patent No. 6,737,056B1 and U.S. patent application publication No. 2004/0132101a1, which disclose Fc mutations that optimize binding of antibodies to Fc receptors.

In certain anti-SEMA 4D binding molecules provided herein, e.g., antibodies or fragments, variants or derivatives thereof, the Fc portion can be mutated using techniques known in the art to modulate, e.g., increase or decrease, effector function. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can decrease Fc receptor binding to the circulating modified antibody, thereby resulting in increased tumor localization. In other cases, constant region modifications consistent with the present disclosure modulate complement fixation and thus may increase or decrease serum half-life. Other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties, which allows for increased localization due to increased antigen specificity or antibody flexibility. The physiological profile, bioavailability, and other biochemical effects resulting from the modification, such as tumor localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

anti-SEMA 4D binding molecules such as antibodies or fragments thereof provided herein include derivatives, for example, modified by: any type of molecule is covalently attached to the antibody such that the covalent attachment does not prevent the antibody from specifically binding its cognate epitope. By way of example, and not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the like. In addition, the derivative may contain one or more non-canonical amino acids.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain of similar charge. Families of amino acid residues having side chains carrying similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or a portion of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind to an anti-SEMA 4D polypeptide, block the interaction of SEMA4D with its receptor, or inhibit, delay, or reduce metastasis in a subject, such as a cancer patient).

For example, it is possible to introduce mutations only in the framework regions or only in the CDR regions of an antibody molecule. The introduced mutation may be a silent or neutral missense mutation, i.e. having no or little effect on the ability of the antibody to bind antigen. These types of mutations may be useful for optimizing codon usage or for improving antibody production in hybridomas. Alternatively, a non-neutral missense mutation may alter the ability of an antibody to bind to an antigen. One skilled in the art will be able to design and test mutant molecules having desired properties, such as no change in antigen binding activity or a change in binding activity (e.g., an improvement in antigen binding activity or a change in antibody specificity). Following mutagenesis, the encoded protein may be expressed in a conventional manner, and the functional activity and/or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to at least one epitope of the SEMA4D polypeptide) may be determined using the techniques described herein or by modifying the techniques known in the art in a conventional manner.

In certain aspects, an anti-SEMA 4D binding molecule, e.g., an antibody or fragment thereof, provided herein can comprise at least one optimized Complementarity Determining Region (CDR). By "optimized CDR," it is meant that the CDR has been modified and optimized to improve the binding affinity and/or anti-SEMA 4D activity conferred on an anti-SEMA 4D binding molecule comprising the optimized CDR. An "anti-SEMA 4D activity" or "SEMA 4D blocking activity" may include an activity that modulates, e.g., reduces, eliminates, reduces or prevents, one or more SEMA4D activities. Non-limiting SEMA4D activities include: b cell activation, aggregation and survival; CD 40-induced proliferation and antibody production; antibody responses to T cell-dependent antigens; t cell or other immune cell proliferation; maturation of dendritic cells; demyelination and axonal degeneration; apoptosis of multipotent neural precursors and/or oligodendrocytes; inducing endothelial cell migration; inhibiting spontaneous monocyte migration; promote tumor cell growth or metastasis, bind cell surface plexin-B1 or other receptors, or any other activity associated with soluble SEMA4D or SEMA4D expressed on the surface of SEMA4D + cells. In a particular embodiment, anti-SEMA 4D activity comprises the ability to inhibit, delay or reduce tumor metastasis in combination with or independently of primary tumor cell growth and tumor metastasis. anti-SEMA 4D activity may also be attributed to a reduction in the occurrence or severity of diseases associated with SEMA4D expression, including but not limited to certain types of cancer, including solid tumors and lymphomas; autoimmune diseases; inflammatory diseases, including Central Nervous System (CNS) and Peripheral Nervous System (PNS) inflammatory diseases; neurodegenerative diseases; graft rejection and invasive angiogenesis.

Polynucleotides encoding anti-SEMA 4D antibodies

The present disclosure also provides an isolated polynucleotide or two or more polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein.

In one aspect, the present disclosure provides an isolated polynucleotide or polynucleotide combination comprising one or more nucleic acid sequences encoding an anti-SEMA 4D binding molecule as provided herein, e.g., an antibody or fragment, variant or derivative thereof or a subunit thereof, e.g., a heavy chain subunit or fragment thereof or a light chain subunit or fragment thereof.

In certain aspects, a polynucleotide or combination of polynucleotides as provided herein comprises a nucleic acid sequence encoding a VH of an anti-SEMA 4D binding molecule (e.g., an antibody or fragment, variant, or derivative thereof). In certain aspects, a polynucleotide or combination of polynucleotides as provided herein comprises a nucleic acid sequence encoding the VL of an anti-SEMA 4D binding molecule (e.g., an antibody or fragment, variant, or derivative thereof). In certain aspects, a polynucleotide or combination of polynucleotides as provided herein comprises a nucleic acid sequence encoding the VH of an anti-SEMA 4D binding molecule (e.g., an antibody or fragment, variant, or derivative thereof) and a nucleic acid sequence encoding the VL of an anti-SEMA 4D binding molecule, e.g., an antibody or fragment, variant, or derivative thereof. In certain aspects, the VH encoding nucleic acid sequence and the VL encoding nucleic acid sequence are located on the same vector. Such vectors are provided by the present disclosure. In certain aspects, the VH encoding nucleic acid sequence and the VL encoding nucleic acid sequence are located on separate vectors. The vectors are also provided by the present disclosure. The vectors provided by the present disclosure may further comprise genetic elements to allow expression of the antibody or fragment thereof. Such genetic elements as promoters, polyadenylation sequences and enhancers are described elsewhere herein. The present disclosure further provides a host cell comprising a polynucleotide or combination of polynucleotides provided herein and/or one or more vectors provided herein.

Also provided is a method for producing an anti-SEMA 4D antibody or fragment, variant or derivative thereof as provided herein, wherein the method comprises culturing a host cell as provided herein and recovering the antibody or fragment thereof.

In certain aspects, the present disclosure provides an isolated polynucleotide or two or more polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA 4D antibody or antigen-binding fragment, variant or derivative thereof or subunit thereof, wherein the antibody comprises a VH comprising complementarity determining regions (HCDR) HCDR1, HCDR2 and HCDR3, and a VL comprising amino acid sequences identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, or identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, except for one, two, three or four amino acid substitutions in one, two or all three of the HCDRs; and the VL comprises complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences identical to SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, or identical to SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, except for one, two, three, or four amino acid substitutions in one, two, or all three of the LCDRs.

In certain aspects, the present disclosure provides an isolated polynucleotide or two or more polynucleotides comprising one or more nucleic acid sequences encoding an anti-SEMA 4D antibody or antigen-binding fragment, variant, or derivative thereof, or subunit thereof, wherein the antibody comprises a VH comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No. 1 and a VL comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No. 5.

Any of the above polynucleotides may further include additional nucleic acid sequences encoding, for example, a heavy or light chain constant region or fragment thereof as described elsewhere herein, a signal peptide to direct secretion of the encoded polypeptide, or other heterologous polypeptides as described herein. Furthermore, as described in more detail elsewhere herein, the present disclosure includes compositions comprising one or more of the foregoing polynucleotides.

In one embodiment, the disclosure includes a composition comprising a first polynucleotide comprising a nucleic acid sequence encoding a VH as described herein and a second polynucleotide encoding a VL as described herein.

The present disclosure also includes fragments of the polynucleotides provided herein, as described elsewhere. In addition, polynucleotides encoding fusion polypeptides, Fab fragments, and other derivatives as described herein are provided.

Polynucleotides provided by the present disclosure may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, then a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, Bio Techniques 17:242 (1994)), which, in brief, involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, followed by amplifying the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be produced from a nucleic acid sequence derived from a suitable source. If clones containing polynucleotides encoding a particular antibody are not available, but the sequence of the antibody molecule is known, then the nucleic acid encoding the antibody can be synthesized chemically, or from a suitable source (e.g., an antibody cDNA library, or from a nucleic acid such as a poly A)⁺RNA-generated cDNA library, isolated from any tissue or cell expressing an antibody or other anti-SEMA 4D antibody such as hybridoma cells selected to express the antibody): PCR amplification using synthetic primers hybridizable to the 3 'and 5' ends of the sequence or cloning using oligonucleotide probes specific for the particular gene sequence to be identified, e.g., a cDNA clone from a cDNA library encoding an antibody or other anti-SEMA 4D antibody. Any method well known in the art may then be used to clone the amplified nucleic acid produced by PCR into a replicable cloning vector.

Once the nucleotide sequence and corresponding amino acid sequence of the anti-SEMA 4D antibody, or antigen-binding fragment, variant, or derivative thereof, are determined, its nucleotide sequence can be manipulated to generate antibodies having different amino acid sequences, e.g., to create amino acid substitutions, deletions, and/or insertions, using methods well known in the art for manipulating nucleotide sequences, e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., Sambrook et al (1990) Molecular Cloning, A Laboratory Manual (2 nd edition; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and techniques described in Ausubel et al (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY), which are all incorporated herein by reference in their entirety).

A polynucleotide or combination of polynucleotides encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) can consist of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide or combination of polynucleotides encoding an anti-SEMA 4D antibody or antigen-binding fragment, variant or derivative thereof may consist of single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single-and double-stranded regions. Furthermore, a polynucleotide or combination of polynucleotides encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) can be composed of a triple-stranded region comprising RNA or DNA or both RNA and DNA. A polynucleotide or combination of polynucleotides encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms.

An isolated polynucleotide or combination of polynucleotides encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by: one or more nucleotide substitutions, additions or deletions are introduced into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. In certain aspects, conservative amino acid substitutions may be made at one or more nonessential amino acid residues.

Fusion proteins and antibody conjugates

As discussed in more detail elsewhere herein, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be further recombinantly fused at the N-terminus or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other heterologous moiety. For example, anti-SEMA 4D antibodies can be recombinantly fused or conjugated to molecules suitable for use as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. patent nos. 5,314,995; and EP 396,387. In certain aspects, the heterologous moiety can be a polypeptide, cytotoxic agent, therapeutic agent, prodrug, lipid, carbohydrate, nucleic acid, detectable label, polymer, or any combination thereof. Exemplary heterologous polypeptides include, without limitation, binding molecules, enzymes, cytokines, lymphokines, hormone peptides, or any combination thereof. Exemplary cytotoxic agents include, without limitation, radionuclides, biotoxins, enzymatically active toxins, or any combination thereof. Exemplary detectable labels include, without limitation, enzymes, fluorescent labels, chemiluminescent labels, bioluminescent labels, radioactive labels, or any combination thereof.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can include a derivative that is modified, for example, by: any type of moiety is covalently attached to the antibody such that the covalent attachment does not prevent the binding molecule from binding to SEMA 4D. By way of example, and not by way of limitation, antibody derivatives may be modified, for example, by: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the like. In addition, the derivative may contain one or more non-canonical amino acids.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may be composed of amino acids joined to one another by peptide bonds or modified peptide bonds, such as peptide isosteres, and may contain amino acids in addition to the 20 gene-encoded amino acids. For example, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may be present anywhere in the anti-SEMA 4D binding molecule, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini, or on moieties such as carbohydrates. Furthermore, a given anti-SEMA 4D binding molecule may contain many types of modifications. anti-SEMA 4D binding molecules may be branched, for example due to ubiquitination, and they may be circular with or without branching. Cyclic, branched, and branched cyclic anti-SEMA 4D binding molecules may result from post-translational natural processes, or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, transfer RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination. (see, e.g., Proteins- -structures and molecular Properties, T.E.Creighton, W.H.Freeman and Company, NY; 2 nd edition (1993); Johnson eds (1983) Posttranslation compatibility Modification of Proteins (academic Press, NY), pages 1-12; Seifter et al, meth.enzymol.182: 626-.

The present disclosure also provides fusion proteins comprising an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused may be suitable for functioning or for targeting against a cell expressing the SEMA4D polypeptide. An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be fused or conjugated to one or more heterologous polypeptides or other moieties using methods known in the art to increase the in vivo half-life of the polypeptide or used in immunoassays. For example, in one embodiment, PEG or human serum albumin can be fused or conjugated to an anti-SEMA 4D binding molecule as provided herein to increase their in vivo half-life. See Leong et al, Cytokine 16:106 (2001); adv.in drug Deliv.Rev.54:531 (2002); or Weir et al, biochem. Soc. transactions 30:512 (2002).

In addition, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be fused to one or more marker sequences, such as peptides, to facilitate their purification or detection. In certain embodiments, the tag amino acid sequence is a hexa-histidine peptide, such as a tag provided in the pQE vector (QIAGEN, Inc.), as well as other tag amino acid sequences, many of which are commercially available. As described in Gentz et al, Proc.Natl.Acad.Sci.USA86:821-824(1989), for example, hexahistidine provides convenient purification of the fusion protein. Other peptide tags suitable for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:767(1984)), and the "flag" tag.

Fusion proteins can be prepared using methods well known in the art (see, e.g., U.S. Pat. nos. 5,116,964 and 5,225,538). The precise site at which the fusion is performed can be empirically chosen to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell to achieve expression.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen binding fragment, variant, or derivative thereof) as provided herein can be used in unconjugated form, or can be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, aid in target detection, or for use in imaging or therapy of a patient. An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be labeled or conjugated before or after purification, or when subjected to purification.

In particular, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be conjugated to a therapeutic agent, prodrug, cytotoxic agent, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

One skilled in the art will appreciate that conjugates can also be assembled using a variety of techniques, depending on the reagent selected for conjugation. For example, conjugates with biotin are prepared, for example, by reacting a binding polypeptide with an activated ester of biotin, such as biotin N-hydroxysuccinimide ester. Similarly, conjugates with fluorescent labels can be prepared in the presence of a coupling agent such as those listed herein, or by reaction with an isothiocyanate, such as fluorescein isothiocyanate. Conjugates of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be prepared in a similar manner.

The present disclosure further encompasses anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein conjugated to a diagnostic or therapeutic agent. The anti-SEMA 4D binding molecules can be used diagnostically, e.g., to monitor the development or progression of a disease, e.g., to determine the efficacy of a given therapeutic and/or prophylactic regimen, as part of a clinical testing procedure. For example, detection may be facilitated by coupling an anti-SEMA 4D binding molecule to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals in the case of various positron emission tomography, and nonradioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents according to the present disclosure, see, e.g., U.S. Pat. No. 4,741,900. Examples of aptamers include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine (rhodamine), dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin (R)phycerythrin); an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include¹²⁵I、¹³¹I、¹¹¹In、⁹⁰Y is or⁹⁹Tc。

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be conjugated to a therapeutic moiety such as a cytotoxin, therapeutic agent, or radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is harmful to a cell.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged anti-SEMA 4D binding molecule is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly suitable chemiluminescent labeling compounds are luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.

One way in which an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant or derivative thereof) as provided herein may be detectably labeled is by linking The anti-SEMA 4D binding molecule to an Enzyme and using The Linked product in an Enzyme Immunoassay (EIA) (Voller, A., "The Enzyme Linked immunological assay (E LISA)" Microbiological Association query Publication, Walkersvill, Md.; Diagnostic Horizons 2:1-7 (1978); Voller et al, J.Clin. Pathol.31:507-520 (1978); Butler, meth.Enzyl.73: 482-; 523 (1981); Ma gg editor (1980) edition Enzyme, Shewa et al, Inc.; Yeast et al, CPwa et al, Crypthecox et al, Tokyu et al, Tokyo et al, Kyoya, 1981). An enzyme that binds to an anti-SEMA 4D binding molecule will react with an appropriate substrate, e.g., a chromogenic substrate, in a manner that produces a chemical moiety that can be detected, for example, by spectrophotometric means, fluorometric means, or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalytic enzyme, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Alternatively, detection can be achieved by colorimetric methods employing chromogenic substrates for the enzyme. Detection can also be achieved by visually comparing the extent of enzymatic reaction of the substrate as compared to a similarly prepared standard.

Detection can also be accomplished using any of a variety of other immunoassays. For example, by radiolabelling an anti-SEMA 4D binding molecule, it is possible to detect the binding molecule by using Radioimmunoassay (RIA). The radioisotope may be detected by means including, but not limited to, gamma counter, scintillation counter, or autoradiography.

anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein may also use fluorescent emitting metals such as¹⁵²Eu or other metals of the lanthanide series to be detectably labeled. These metals can be attached to the binding molecule using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various moieties to anti-SEMA 4D binding molecules as provided herein are well known to those of ordinary skill in the art.

Expression of antibody polypeptides

DNA sequences encoding the light and heavy chains of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be prepared simultaneously or separately using reverse transcriptase and a DNA polymerase according to well-known methods. PCR is initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be screened by consensus primers or larger homology probes such as mouse constant region probes.

DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restricted localization and sequencing according to standard well-known techniques, for example as detailed in references related to recombinant DNA techniques provided elsewhere herein.

After manipulation of the isolated genetic material to provide an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein, the polynucleotide encoding the binding molecule can be inserted into an expression vector for introduction into one or more host cells that can be used to produce a desired amount of anti-SEMA 4D binding molecule.

Recombinant expression of an anti-SEMA 4D antibody or fragment, derivative or analogue thereof, e.g. the heavy or light chain of an antibody, requires the construction of one or more expression vectors containing a polynucleotide or combination of polynucleotides encoding the antibody. Once a polynucleotide or combination of polynucleotides encoding an antibody molecule or heavy or light chain of an antibody or portion thereof (e.g., a portion comprising a heavy chain variable region or a light chain variable region) has been obtained, one or more vectors for producing the antibody molecule can be produced by recombinant DNA techniques, using techniques well known in the art. Thus, described herein are methods for producing a protein by expressing a polynucleotide or combination of polynucleotides comprising antibody-encoding nucleic acid sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present disclosure provides a replicable vector comprising a nucleic acid sequence encoding an anti-SEMA 4D binding molecule as provided herein, e.g., an antibody or a heavy or light chain thereof or a variable region of a heavy or light chain thereof, operably linked to a promoter. The vector may include nucleotide sequences encoding the constant regions of the heavy and/or light chains of the antibody (see, e.g., PCT publication WO 86/05807; PCT publication WO 89/01036; and U.S. Pat. No. 5,122,464), and the variable regions of the antibody may be cloned into such a vector to express the entire heavy or light chain.

The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene in a host cell. The vector can be readily selected from, for example, plasmids, phages and viruses such as retroviruses, as known to those skilled in the art. Generally, vectors compatible with the present disclosure will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and be capable of entry into and/or replication in eukaryotic or prokaryotic cells.

Numerous expression vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites (IRES). In addition, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. The marker may provide the original auxotrophy of the auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

In certain aspects, the cloned variable region nucleic acid molecule can be inserted into an expression vector along with the heavy and light chain constant region genes synthesized as discussed above. Of course, any expression vector capable of eliciting expression in eukaryotic cells can be used. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF 1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1 and pZeoSV2 (available from Invitrogen, San Diego, Calif.) and plasmid pCI (available from Promega, Madison, Wis.). In general, screening of a large number of transformed cells for those expressing suitable high levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by a robotic system.

More generally, once a vector or DNA sequence encoding a monomeric subunit of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with whole virus. See Ridgway (1988) "mammalianExpression Vectors", Vectors, Rodriguez and Denhardt eds (Butterworks, Boston, Mass.), Chapter 24.2, page 470-472. Typically, introduction of the plasmid into the host is performed by electroporation. Host cells with the expression constructs can be grown under conditions suitable for production of light and heavy chains, and heavy and/or light chain protein synthesis and assembly assayed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS), immunohistochemical analysis, and the like.

The expression vector can be transferred into a host cell by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein. Accordingly, the disclosure includes host cells comprising a polynucleotide or combination of polynucleotides encoding an antibody or heavy or light chain thereof as provided herein operably linked to a promoter, e.g., a heterologous promoter. In certain embodiments regarding the expression of diabodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell to express and assemble the entire binding molecule, as described in detail below.

As used herein, "host cell" refers to a cell having a vector constructed using recombinant DNA techniques and encoding at least one heterologous polynucleotide. In describing methods for isolating antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody unless explicitly specified otherwise. In other words, recovery of the polypeptide from "cells" can mean from a brief centrifugation of whole cells or from a cell culture containing both culture medium and suspension cells.

A variety of host expression vector systems may be used to express the anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein. The host expression system represents a vehicle through which the coding sequence of interest can be produced and subsequently purified, but also represents a cell that can express an antibody molecule as provided herein in situ when transformed or transfected with the appropriate nucleotide coding sequence. These expression systems include, but are not limited to, microorganisms such as bacteria (e.g., escherichia coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., yeast (Saccharomyces), Pichia (Pichia)) transformed with a recombinant yeast expression vector containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) having recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter or the human cytomegalovirus immediate early promoter). In certain aspects, especially for expression of intact recombinant antibody molecules, bacterial cells such as E.coli or eukaryotic cells can be used to express the recombinant antibody molecule. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells in combination with vectors are an efficient antibody expression system (Foecking et al, Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).

The host cell line for protein expression may be of mammalian origin. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11(DHFR deficient Chinese hamster ovary strain), HeLa (human cervical cancer), CVI (monkey kidney strain), COS (derivative of CVI with the SV 40T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney strain), SP2/O (mouse myeloma), P3X 63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). Host cell lines are generally available from commercial service, the American tissue culture Collection, or from the open literature.

In addition, host cell lines can be selected that modulate the expression of the inserted sequences or modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with the cellular machinery for achieving the appropriate primary transcript processing, glycosylation and phosphorylation of the gene product can be used.

For long-term high-yield production of recombinant proteins, stable expression can be used. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of the foreign DNA, the engineered cells can be grown in an enrichment medium for 1-2 days, followed by a switch to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that stably express the antibody molecule.

Expression levels of antibody molecules can be increased by vector Amplification (for review, see Bebbington and Hentschel (1987) "The Use of Vectors Based on Gene Amplification for The expression of Cloned Genes in Mammalian Cells in DNA Cloning" (Academic Press, NY) volume 3). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will result in an increase in the copy number of the marker gene. Because the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al, mol. cell. biol.3:257 (1983)).

In vitro production allows scaling up to produce large quantities of the desired polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or in a continuous stirrer reactor, or fixed or embedded cell culture on, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and/or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography over DEAE-cellulose or (immuno) affinity chromatography, for example after biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein.

A nucleic acid molecule encoding an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can also be expressed in a non-mammalian cell, such as an insect, bacterial, or yeast or plant cell. Bacteria susceptible to uptake of nucleic acids include members of the family enterobacteriaceae (enterobacteriaceae), such as strains of escherichia coli or Salmonella (Salmonella); and members of the Bacillus family (Bacillus), such as Bacillus subtilis; a member of the genus Pneumococcus (Pneumococcus); members of the genus Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). It will be further appreciated that when expressed in bacteria, the heterologous polypeptide typically becomes part of an inclusion body. Inclusion bodies can be isolated, purified, and then assembled into functional molecules. When a tetravalent form of the antibody is desired, the subunits can then self-assemble into a tetravalent antibody (WO 02/096948a 2).

In the case of bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large amounts of such proteins are to be produced to produce pharmaceutical compositions of antibody molecules, vectors that direct high levels of expression of the fusion protein product that are readily purified may be desirable.

In addition to prokaryotes, eukaryotic microorganisms may also be used. Among eukaryotic microorganisms, Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common baker's yeast is the most commonly used, but many other strains are commonly available, such as Pichia pastoris (Pichia pastoris).

In the case of insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is commonly used as a vector for the expression of foreign genes. The virus was grown in Spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be individually cloned into nonessential regions of the virus, such as the polyhedrin gene, and placed under the control of an AcNPV promoter, such as the polyhedrin promoter.

Once the anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein has been recombinantly expressed, it can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly using affinity for protein a, and size fractionation column chromatography), centrifugation, differential solubilization, or by any other standard technique for purifying proteins. Alternatively, methods for increasing the affinity of an antibody as provided herein are disclosed in U.S. patent application publication No. 20020123057 a 1.

Methods of treatment using therapeutic anti-SEMA 4D antibodies

The present disclosure provides methods for treating a subject having a disease or disorder associated with a pathology of SEMA4D using an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein.

The following discussion relates to diagnostic methods and treatments for various diseases and conditions using anti-SEMA 4D binding molecules (e.g., antibodies or antigen binding fragments, variants or derivatives thereof) as provided herein, e.g., capable of specifically binding to SEMA4D, e.g., human SEMA4D, mouse SEMA4D, or human SEMA4D and mouse SEMA4D, and having SEMA4D neutralizing activity.

In one aspect, the present disclosure provides a method for neutralizing SEMA4D in a human subject, comprising administering to the human subject an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein. In certain aspects, the human subject is in need of treatment for an autoimmune disease or disorder, an inflammatory disease or disorder, cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof. In certain aspects, the neuroinflammatory disease or disorder is multiple sclerosis. In certain aspects, the neurodegenerative disease or disorder is stroke, alzheimer's disease, parkinson's disease, huntington's disease, down syndrome, ataxia, Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), HIV-associated cognitive impairment, CNS lupus, mild cognitive impairment, or a combination thereof. In certain aspects, the autoimmune or inflammatory disease is arthritis, such as rheumatoid arthritis, atherosclerosis (see, e.g., PCT publication No. WO2015/054628, which is incorporated herein by reference in its entirety), or a bone degenerative disease such as osteoporosis (see, e.g., U.S. patent No. 9,447,191, which is incorporated herein by reference in its entirety).

In one aspect, treatment comprises applying or administering to a subject an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein, to an isolated tissue or cell line from a subject, wherein the subject has a disease, a symptom of a disease, or has a predisposition toward a disease. In another aspect, treatment also includes applying or administering to a subject or to an isolated tissue or cell line from a patient having a disease, a symptom of a disease, or a predisposition toward a disease, a pharmaceutical composition comprising an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein.

In one aspect, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may be useful for treating cancer, e.g., treating various malignant and non-malignant tumors. By "antineoplastic activity" it is meant a reduction in the rate of proliferation or accumulation of malignant cells, thus, a reduction in the growth rate of existing tumors or in the case of tumors that appear during therapy, and/or the destruction of existing neoplastic (tumor) cells or newly forming neoplastic cells, thus, a reduction in the overall size of the tumor during therapy. For example, therapy with an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may elicit a physiological response, such as decreased angiogenesis or migration of CTLs into the tumor microenvironment. Methods for treating cancer with an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be found, for example, in PCT publication No. WO 2014/209802[ combination immunotherapy ], which is incorporated herein by reference in its entirety.

In one aspect, the present disclosure provides an anti-SEMA 4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, as provided herein for use as a medicament, particularly for the treatment or prophylaxis of cancer or for use in the context of a precancerous condition or lesion.

In another aspect, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may be useful for treating an autoimmune disease or an inflammatory disease. In another aspect, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may be useful for treating a neuroinflammatory or neurodegenerative disease. Methods for treating neuroinflammatory or neurodegenerative diseases can be found, for example, in PCT publication nos. WO2013/055922[ BBB ], WO2015/061330[ HD ], and WO 2013/170221[ neurogenesis/stroke ], the contents of which are incorporated by reference herein in their entirety.

According to the methods provided herein, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used to promote a positive therapeutic response with respect to malignant human cells. By "positive therapeutic response" with respect to cancer treatment, it is meant an improvement in the disease associated with the anti-tumor activity of these binding molecules, e.g., antibodies or fragments thereof, and/or an improvement in the symptoms associated with the disease. That is, an anti-proliferative effect, prevention of further tumor outgrowth, reduction in tumor size, reduction in tumor vasculature, reduction in the number of cancer cells, and/or reduction in one or more symptoms associated with the disease can be observed. Thus, for example, improvement in a disease can be characterized as a complete response. By "complete response" it is meant the absence of a clinically detectable disease, with any prior abnormal radiographic studies, normalization of bone marrow and cerebrospinal fluid (CSF). Alternatively, improvement in the disease may be classified as partial response. By "partial response" it is meant that all measurable tumor burden (i.e., the number of tumor cells present in the subject) is reduced by at least about 50% in the absence of new lesions and persists for at least one month. This response is only applicable to measurable tumors.

anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein may also be useful for treating inflammatory diseases and deficiencies or conditions of the immune system. Inflammatory diseases are characterized by inflammation and tissue destruction, or a combination thereof. By "anti-inflammatory activity" it is meant reducing or preventing inflammation. "inflammatory disease" includes any inflammatory immune-mediated process in which the priming event or target of an immune response involves a non-self antigen, including, for example, an alloantigen, a xenoantigen, a viral antigen, a bacterial antigen, an unknown antigen, or an allergen. In one embodiment, the inflammatory disease is an inflammatory disorder of the peripheral or central nervous system. In another embodiment, the inflammatory disease is an inflammatory disorder of the joints.

Furthermore, the term "inflammatory disease" includes "autoimmune disease". As used herein, the term "autoimmunity" is generally understood to encompass inflammatory immune-mediated processes involving "self" antigens. In autoimmune diseases, autoantigens trigger a host immune response. Autoimmune diseases can result from inappropriate immune responses against self-antigens (autoantigens) that deviate from the normal state of self-tolerance. In general, antibodies (particularly but not exclusively IgG antibodies) that act as cytotoxic molecules or immune complexes are the primary mediators of various autoimmune diseases, many of which can be debilitating or life threatening.

In one embodiment, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used to treat Multiple Sclerosis (MS). MS, also known as disseminated sclerosis or disseminated encephalomyelitis, is an autoimmune disorder in which the immune system attacks the central nervous system, leading to demyelination. The name multiple sclerosis relates to scars (sclerosis, also known as plaques or lesions) formed in the nervous system. MS lesions typically involve white matter areas of the ventricles of the brain, the brainstem, the basal ganglia and the spinal cord, as well as the optic nerve, close to the cerebellum. MS results in the destruction of oligodendrocytes, which are the cells responsible for the creation and maintenance of myelin sheaths. MS results in thinning or complete loss of myelin and, as the disease progresses, axonal transection.

Neurological symptoms can vary with MS, and the disease often progresses to physical and cognitive disability. MS takes several forms, with new symptoms appearing in discrete episodes (recurrent form) or accumulating slowly over time (progressive form). Between episodes, symptoms may go away completely, but permanent nerve damage often results, especially as the disease progresses.

Neutralization of SEMA4D using an anti-SEMA 4D binding molecule (e.g., an antibody or antigen binding fragment, variant, or derivative thereof) as provided herein can be used to reduce the severity of MS by several different mechanisms, e.g., an anti-SEMA 4D monoclonal antibody can block immune maturation and activation by SEMA4D to reduce the rate of relapse by reducing secondary immune responses to CNS antigens, and an anti-SEMA 4D monoclonal antibody can block the effect of soluble SEMA4D in mediating apoptosis of oligodendrocytes in the CNS, can reduce disease severity by reducing demyelination.

In one aspect, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used to treat arthritis. Arthritis is an inflammatory disease of the joints that can be caused by autoimmune disorders in which the immune system attacks the joints. In certain embodiments, the arthritis is selected from the group consisting of: osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, rheumatoid arthritis, juvenile-onset rheumatoid arthritis, infectious arthritis, inflammatory arthritis, septic arthritis, degenerative arthritis, destructive arthritis, and Lyme arthritis (Lyme arthritis). In one embodiment, the arthritis is Rheumatoid Arthritis (RA).

The present disclosure includes methods of treating or preventing arthritis by administering to a subject an anti-SEMA 4D binding molecule as provided herein. The methods as provided herein can reduce pain, swelling, or stiffness associated with arthritis, such as rheumatoid arthritis. The present disclosure also relates to methods for improving joint performance, function, and health. In some embodiments of the disclosure, treatment results in a decrease in the severity score of arthritis, a decrease in the severity/area under the curve of arthritis, a decrease in histopathological parameters associated with arthritis (inflammation, pannus, cartilage damage, and bone damage), a decrease in serum arachidonic acid levels, or a decrease in anti-collagen antibodies. In certain embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms associated with arthritis; prevention of arthritis; delay in the onset of arthritis; a reduced incidence of arthritis in the population; a reduced extent of arthritis-associated conditions; stabilization (e.g., not worsening) of the state of a disorder, condition, or disease associated with arthritis; delay in the onset or slowing of progression of a condition, disorder or disease associated with arthritis; an improvement in a condition, disorder or disease state, an amelioration (whether partial or total) of a condition, disorder or disease associated with arthritis, whether detectable or undetectable; or an improvement or amelioration of a condition, disorder or disease associated with arthritis.

The methods provided herein can be used to treat an individual having arthritis or at risk of developing arthritis. Accordingly, in some embodiments, the present disclosure provides a method of treating a subject having a normal joint, a borderline arthritic joint, or a genuine arthritic joint, the method comprising administering to a subject as described herein an anti-SEMA 4D binding molecule as provided herein. In some embodiments, the methods provided herein can be used to treat chronic arthritis for the remainder of the subject's life.

According to the methods as provided herein, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used to facilitate a positive therapeutic response with respect to treating or preventing an autoimmune disease and/or an inflammatory disease. By "positive therapeutic response" with respect to autoimmune and/or inflammatory diseases, it is meant an improvement in the disease associated with the anti-inflammatory activity, anti-angiogenic activity, anti-apoptotic activity, etc., of these antibodies, and/or an improvement in the symptoms associated with the disease. That is, an antiproliferative effect, prevention of further proliferation of SEMA4D expressing cells, reduction of inflammatory responses including, but not limited to, inflammatory cytokines, adhesion molecules, proteases, immunoglobulins (in the case where the cell carrying SEMA4D is a B cell), reduced secretion of combinations thereof, etc., increased production of anti-inflammatory proteins, reduced number of autoreactive cells, increased immune tolerance, inhibition of autoreactive cell survival, reduced apoptosis, reduced endothelial cell migration, increased spontaneous monocyte migration, reduction and/or reduction of one or more symptoms mediated by stimulation of SEMA4D or SEMA4D expressing cells may be observed. The positive therapeutic response is not limited to the route of administration and may include administration to the donor, donor tissue (such as, for example, organ perfusion), host, any combination thereof, and the like.

Clinical responses can be assessed using screening techniques such as Magnetic Resonance Imaging (MRI) scans, x-ray radiographic imaging, Computed Tomography (CT) scans, flow cytometry or Fluorescence Activated Cell Sorter (FACS) analysis, histological analysis, macropathological analysis, and blood chemistry analysis, including but not limited to detection of changes by ELISA, RIA, chromatography, and the like. In addition to these positive therapeutic responses, a subject undergoing therapy with an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein may experience a beneficial effect in which symptoms associated with the disease are ameliorated.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used in combination with at least one other cancer therapy, including but not limited to surgery or surgical procedures; immunomodulatory therapy, radiotherapy; chemotherapy, optionally in combination with autologous bone marrow transplantation, or other cancer therapies; wherein the additional cancer therapy is administered before, during or after anti-SEMA 4D binding molecule, e.g., antibody or antigen binding fragment thereof therapy. Thus, when combination therapy includes administration of an anti-SEMA 4D binding molecule, e.g., an antibody or antigen-binding fragment thereof, as provided herein in combination with administration of another therapeutic agent as chemotherapy, radiation therapy, other anti-cancer antibody therapies, small molecule-based cancer therapies, or vaccine/immunotherapy-based cancer therapies, the methods as provided herein encompass co-administration, use of separate or single pharmaceutical formulations, or/and sequential administration in either order.

The anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein can be used in combination with any known therapy for autoimmune and inflammatory diseases, including any agent or combination of agents known to be suitable for, or used or currently used for the treatment of autoimmune and inflammatory diseases. Thus, where combination therapy includes administration of an anti-SEMA 4D binding molecule in combination with administration of another therapeutic agent, the methods provided herein encompass co-administration, use of separate or single pharmaceutical formulations, and sequential administration in either order. In some embodiments, the anti-SEMA 4D antibodies described herein are administered in combination with an immunosuppressive drug or an anti-inflammatory drug, wherein the antibody and therapeutic agent may be administered sequentially in either order, or simultaneously (i.e., concurrently or within the same time frame).

In certain aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used alone, or in combination with an immunosuppressive drug, to treat and/or prevent rheumatoid arthritis. As discussed above, treatment effectiveness may be assessed using any means, and includes, but is not limited to, effectiveness as measured by clinical response determined according to American College of Rheumatology (American College of Rheumatology) guidelines, European league of Rheumatology (European league of Rheumatology) guidelines, or any other criteria. See, e.g., Felson et al, Arthritis Rheum.38:727-35(1995) and van Gestel et al, Arthritis Rheum.39:34-40 (1996).

In other aspects, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used alone, or in combination with an immunosuppressive drug, to treat and/or prevent multiple sclerosis.

Another aspect of the present disclosure is the use of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein for diagnostic monitoring of protein levels in a tissue, e.g., to determine the efficacy of a given therapeutic regimen, as part of a clinical testing procedure. For example, detection may be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of aptamers include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include¹²⁵I、¹³¹I、³⁵S or³H。

Pharmaceutical compositions and methods of administration

Methods of making and administering to a subject in need thereof an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein are well known to, or readily determined by, those skilled in the art. The route of administration of the anti-SEMA 4D binding molecule may be, for example, oral, parenteral, by inhalation, or via a surface. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although all such administration forms are contemplated as being within the scope of the present disclosure, an example of an administration form would be a solution for injection, particularly for intravenous or intra-arterial injection or instillation. Typically, a pharmaceutical composition suitable for injection may comprise a buffer (e.g., an acetate, phosphate or citrate buffer), a surfactant (e.g., a polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods compatible with the teachings herein, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be delivered directly to the site of an adverse condition, such as a solid tumor, thereby increasing exposure of diseased tissue to the therapeutic agent.

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be administered in a pharmaceutically effective amount to treat SEMA 4D-mediated diseases in vivo, such as cancer, autoimmune diseases, inflammatory diseases, including Central Nervous System (CNS) and Peripheral Nervous System (PNS) inflammatory diseases, neurodegenerative diseases, and aggressive angiogenesis. In this regard, it is to be understood that the disclosed binding molecules can be formulated so as to facilitate administration as well as promote stability of the active agent. In certain aspects, a pharmaceutical composition as provided herein can include a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like. For the purposes of this application, a pharmaceutically effective amount of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein, conjugated or unconjugated, means an amount sufficient to achieve effective binding to the target, as well as to achieve a benefit, such as improving the symptoms of a disease or disorder or detecting a substance or cell.

The pharmaceutical compositions used in the present disclosure comprise a pharmaceutically acceptable carrier including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

Formulations for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M, e.g., 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

In certain aspects, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should have a fluidity to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating agent, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for use in the methods of treatment disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16 th edition (1980).

Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal (thimerosal), and the like. In certain aspects, isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium chloride may be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared by: the active compound, e.g., an anti-SEMA 4D binding molecule as provided herein (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof), alone or in combination with other active agents, is incorporated in the required amount in an appropriate solvent, along with one or a combination of ingredients enumerated herein, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation may include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The injectable formulations are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under sterile conditions. In addition, the formulations may be packaged and sold in kits such as those described in U.S. patent application serial No. 09/259,337. The article of manufacture can have a label or package insert indicating that the related composition is suitable for use in treating a subject suffering from or susceptible to a disease or disorder.

Parenteral formulations may be single bolus doses, infusion or loading bolus doses followed by a maintenance dose. These compositions may be administered at specific fixed or variable intervals, for example once a day, or on an "as needed" basis.

Certain pharmaceutical compositions provided by the present disclosure may be administered orally in acceptable dosage forms including, for example, capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions may also be administered by nasal aerosol or by inhalation. The compositions may be prepared as solutions in saline with benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

The amount of anti-SEMA 4D binding molecule (e.g., antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The compositions may be administered as an infusion in a single dose, in multiple doses, or over a defined period of time. Dosage regimens can also be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response).

An anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be administered to a human or other animal in an amount sufficient to produce a therapeutic effect according to the above-mentioned methods of treatment. The anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein can be administered to humans or other animals in conventional dosage forms prepared by combining the antibodies with conventional pharmaceutically acceptable carriers or diluents according to known techniques. One skilled in the art will recognize that the form and nature of a pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it will be combined, the route of administration, and other well-known variables.

By "therapeutically effective dose or amount" or "effective amount" is meant an amount of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein that, when administered, results in a positive therapeutic response with respect to treating a patient having a disease to be treated.

The use of a composition as provided herein for the treatment of a SEMA 4D-mediated disease such as cancer; autoimmune diseases, such as arthritis, multiple sclerosis; inflammatory diseases, including Central Nervous System (CNS) and Peripheral Nervous System (PNS) inflammatory diseases; neurodegenerative diseases; and aggressive angiogenesis, will vary depending on a number of different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dose can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

The amount of anti-SEMA 4D binding molecule (e.g., antibody or antigen-binding fragment, variant, or derivative thereof) to be administered as provided herein is readily determined by one of ordinary skill in the art without undue experimentation. Factors that influence the mode of administration and the corresponding amount of anti-SEMA 4D binding molecule include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health and physical condition of the individual undergoing therapy. Similarly, the amount of anti-SEMA 4D binding molecule to be administered will depend on the mode of administration and whether the subject will be subjected to a single dose or multiple doses of this agent.

The present disclosure also provides the use of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) to manufacture a medicament for treating an autoimmune disease and/or an inflammatory disease, including, for example, arthritis, multiple sclerosis, CNS and PNS inflammatory diseases, neurodegenerative diseases, or cancer.

The present disclosure also provides for the use of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) of the present disclosure to manufacture a medicament for treating an autoimmune disease and/or an inflammatory disease, including, for example, arthritis, multiple sclerosis, CNS and PNS inflammatory diseases, neurodegenerative diseases, or cancer, wherein the medicament is for use in a subject that has been pre-treated with at least one other therapy. By "pretreated" it is meant that the subject has received one or more other therapies (e.g., has been treated with at least one other cancer therapy) prior to receiving an agent comprising an anti-SEMA 4D binding molecule. "pretreatment" includes a subject having been treated with at least one other therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or even within 1 day prior to initiating treatment with an agent comprising an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein. The subject need not be a responder to a pretreatment with a prior therapy or therapies. Thus, a subject receiving an agent comprising an anti-SEMA 4D binding molecule may have responded to prior therapy, or to one or more of prior therapies when the prior therapy includes multiple therapies, or may have failed to respond to prior therapy, or to one or more of prior therapies when the prior therapy includes multiple therapies (e.g., the cancer is refractory). Examples of other cancer therapies for which a subject may have been pre-treated prior to receiving an agent comprising an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein include, but are not limited to, surgery; radiotherapy; chemotherapy, optionally in combination with autologous bone marrow transplantation; other anti-cancer monoclonal antibody therapies; small molecule-based cancer therapies; vaccine/immunotherapy-based cancer therapies; steroid therapy; other cancer therapies; or any combination thereof.

IX. diagnosis

The present disclosure further provides a diagnostic method applicable during diagnosis of a SEMA 4D-mediated disease, such as certain types of cancer, autoimmune diseases, inflammatory diseases, including, for example, arthritis, multiple sclerosis, Central Nervous System (CNS) and Peripheral Nervous System (PNS) inflammatory diseases, neurodegenerative diseases and invasive angiogenesis, which involves measuring the expression level of SEMA4D protein or transcript in a tissue or other cell or body fluid from an individual and comparing the measured expression level to a standard SEMA4D expression level in a normal tissue or body fluid, whereby an increased expression level compared to the standard value is indicative of a condition.

anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein can be used to determine SEMA4D protein levels in biological samples using classical immunohistological methods known to those of skill in the art (see, e.g., Jalkanen et al, J.cell. biol.101: 976-305 (1985); Jalkanen et al, J.cell biol.105:3087-3096 (1987)). Other antibody-based methods suitable for detecting SEMA4D protein expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, or Western blots. Suitable assays are described in more detail elsewhere herein.

By "determining the expression level of a SEMA4D polypeptide", it is meant qualitatively or quantitatively measuring or estimating the level of a SEMA4D polypeptide in a first biological sample, either directly (e.g., by determining or estimating the absolute protein level) or relatively (e.g., by comparing to the level of a disease-associated polypeptide in a second biological sample). In certain aspects, the level of SEMA4D polypeptide expression in a first biological sample can be measured or estimated and compared to a standard SEMA4D polypeptide level taken from a second biological sample obtained from an individual not suffering from a disorder or determined by averaging levels from a population of individuals not suffering from a disorder. As will be appreciated in the art, once the "standard" SEMA4D polypeptide level is known, it can be reused as a comparison standard.

By "biological sample" it is meant any biological sample obtained from an individual, cell line, tissue culture or other cell source potentially expressing SEMA 4D. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

Immunoassay of X

Immunospecific binding of an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant or derivative thereof) as provided herein can be determined by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein a immunoassays, to name a few. Such assays are conventional and well known in the art (see, e.g., eds. Ausubel et al, (1994) Current protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol.1, which is incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below (but are not intended to be described in a limiting manner).

In addition, an anti-SEMA 4D binding molecule (e.g., an antibody or antigen-binding fragment, variant, or derivative thereof) as provided herein can be used histologically in situ to detect a SEMA4D protein or a conserved variant or peptide fragment thereof, as in an immunofluorescence assay, an immunoelectron microscopy assay, or a non-immunoassay. In situ detection may be achieved by: removing the histological specimen from the patient and applying a labeled anti-SEMA 4D antibody or antigen-binding fragment thereof thereto, e.g., by overlaying the labeled antibody (or fragment) on the biological sample. By using this procedure it is possible to determine not only the presence of the SEMA4D protein or of conserved variants or peptide fragments, but also its distribution in the tissue under investigation. Using the present disclosure, one of ordinary skill will readily recognize that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

The binding activity of a number of given anti-SEMA 4D binding molecules (e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof) as provided herein can be determined according to well-known methods. By employing routine experimentation, one skilled in the art will be able to determine effective and optimal assay conditions for each assay.

There are a number of methods available for measuring the affinity of antibody-antigen interactions, but the methods used to determine the rate constant are relatively trivial. Most methods rely on labeling of antibodies or antigens, which inevitably complicates routine measurements and introduces uncertainty in the number measured.

Such as inSurface Plasmon Resonance (SPR) performed above offers many advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) labeling of the antibody or antigen is not required; (ii) the antibody does not need to be purified in advance, and the cell culture supernatant can be directly used; (iii) real-time measurements that allow rapid semi-quantitative comparison of the interaction of different monoclonal antibodies can be achieved and are sufficient for many assessment purposes; (iv) biospecific surfaces can be regenerated so that a series of different monoclonal antibodies can be readily compared under the same conditions; (v) the analysis procedure is fully automated and a series of extensive measurements can be made without user intervention. BIAappications Handbook, AB edition (reprinted 1998),code BR-1001-86; biatech Handbook, version AB (reprinted 1998),code number BR-1001-84. SPR-based binding studies require one member of a binding pair to be immobilized on a sensor surface. The immobilized binding partner is referred to as a ligand. The binding partner in solution is referred to as the analyte. In some cases, the ligand is indirectly attached to the surface by binding another immobilized molecule, referred to as a capture molecule. The SPR response reflects the change in mass concentration at the detector surface as the analyte binds or dissociates.

Based on SPR, real time as interactions occurThe measurement directly monitors the interaction. The technique is well suited for determining kinetic parameters. Comparative affinity ranking is easy to perform and both kinetic and affinity constantsEither can be obtained from sensorgram data.

When an analyte is injected in discrete pulses across the ligand surface, the resulting sensorgram can be divided into three main phases: (i) the analyte associates with the ligand during sample injection; (ii) an equilibrium or steady state during sample injection, wherein the analyte binding rate is balanced by dissociation from the complex; (iii) the analyte dissociates from the surface during the buffer flow.

The association and dissociation phases provide information about the kinetics of the analyte-ligand interaction (k)_aAnd k_dI.e. the rate of complex formation and dissociation, k_d/k_a＝K_D). The equilibration phase provides information about the affinity of the analyte-ligand interaction (K)_D)。

The BIAevaluation software provides a comprehensive tool for curve fitting using both numerical integration and global fitting algorithms. In the case of suitable analysis of the data, the individual rate constants and affinity constants of the interaction can be simplyAnd (5) exploring and obtaining. The range of affinities that can be measured by this technique is extremely broad, ranging from mM to pM.

Epitope specificity is an important feature of monoclonal antibodies. Compared with conventional techniques using radioimmunoassay, ELISA or other surface adsorption methodsEpitope mapping does not require labeling or purification of antibodies and allows for multi-site specific testing using a series of several monoclonal antibodies. In addition, a large number of analyses can be handled automatically.

Paired binding experiments test the ability of two mabs to bind to the same antigen simultaneously. Mabs directed against individual epitopes will bind independently, while mabs directed against the same or closely related epitopes will interfere with each other's binding. By usingThese binding experiments were performed for simplicity.

For example, a capture molecule may be used to bind a first Mab, followed by the sequential addition of antigen and a second Mab. The sensorgram will reveal (1) how much antigen binds to the first Mab, (2) the extent to which the second Mab binds to the surface-attached antigen, and (3) whether reversing the sequence of the pairwise test would change the results if the second Mab did not bind.

Peptide inhibition is another technique for epitope mapping. This approach can complement paired antibody binding studies and can correlate functional epitopes with structural features when the primary sequence of the antigen is known. The peptides or antigen fragments were tested for inhibition of binding of different mabs to the immobilized antigen. Peptides that interfere with the binding of a given MAb are hypothesized to be structurally related to the epitope defined by that MAb.

Unless otherwise indicated, the present disclosure employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are well described in the literature. See, e.g., code of Sambrook et al (1989) Molecular Cloning A Laboratory Manual (2 nd edition; Cold Spring Harbor Laboratory Press); molecular Cloning, compiled by Sambrook et al (1992) Laboratory Manual, (Cold Springs Harbor Laboratory, NY); glover eds, (1985) DNACloning, volumes I and II; gait, eds (1984) Oligonucleotide Synthesis; mullis et al, U.S. Pat. Nos. 4,683,195; hames and Higgins eds (1984) Nucleic Acid Hybridization; hames And Higgins eds (1984) Transcription And transformation; freshney (1987) Culture Of Animal Cells (Alan r. loss, Inc.); immobilized Cells And Enzymes (IRL Press) (1986); perbal (1984) A Practical Guide To Molecular Cloning; monograph, Methods In Enzymology (Academic Press, inc., n.y.); miller and Calos eds (1987) Gene Transfer Vectors for Mammalian Cells, (Cold Spring Harbor Laboratory); wu et al, Methods Inenzymology, volumes 154 and 155; mayer and Walker eds (1987) Immunochemical Methods In cell and Molecular Biology (Academic Press, London); edited by Weir and Blackwell, (1986) handbook of Experimental Immunology, volumes I-IV; manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1986); and in Ausubel et al (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

The general principles of Antibody Engineering are described in Borrebaeck, eds. (1995) Antibody Engineering (2 nd edition; Oxford Univ.Press). The general principles of Protein Engineering are described in Rickwood et al, eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ.Press, Oxford, Eng.). The general principles of antibody and antibody-hapten binding are set forth in the following: nisonoff (1984) Molecular Immunology (2 nd edition; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, the theory Structure and Function (Chapman and Hall, New York, N.Y.). In addition, standard immunological methods known in the art and not explicitly described can be found, for example, in Current Protocols in immunology, John Wiley & Sons, New York; stits et al eds (1994) Basic and Clinical Immunology (8 th edition; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (1980) Selected Methods in Cellular Immunology (W.H.Freeman and Co., NY) were followed.

Standard reference works illustrating the general principles of Immunology include Current Protocols in Immunology, John Wiley & Sons, New York; klein J., Immunology, The Science of Self-Nonself characterization (John Wiley & Sons, NY (1982)); kennett et al (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological analytes (plenum Press, NY); campbell (1984) "Monoclonal Antibody Technology", laboratory technologies in Biochemistry and Molecular Biology, compiled by Burden et al, (Elsevier, Amsterdam); goldsby et al, eds (2000) Kuby Immunology (4 th edition; W.H.Freeman and Co., NY); roitt et al (2001) Immunology (6 th edition; London: Mosby); abbas et al (2005) Cellular and molecular Immunology (5 th edition; Elsevier Health Sciences Division); kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); sambrook and Russell (2001) Molecular Cloning A Laboratory Manual (Cold Spring Harbor Press); lewis (2003) Genes VIII (Prentice Hall, 2003); harlow and Lane (1988) Antibodies A laboratory Manual (Cold Spring Harbor Press); dieffenbach and Dveksler (2003) PCR Primer (Cold spring Harbor Press).

All references cited above and all references cited herein are incorporated by reference in their entirety.

The following examples are provided by way of illustration and not by way of limitation.

Examples

Example 1: production of fully human anti-SEMA 4D monoclonal antibody MAb D2517

Fully human anti-SEMA 4D monoclonal antibody D2517 was generated by the following method. A library of fully human antibodies was generated in vaccinia virus and screened for binding to SEMA4D according to the methods described in U.S. patent application publication No. 2013-0288927-a1, which is incorporated herein by reference in its entirety. A total of 96 antibodies were partitioned into six different epitope groups according to competition ELISA. The competitive binding of human antibodies to the native form of human SEMA4D on Jurkat cells was evaluated using the mouse antibodies mAb76 and mAb67, and a mouse IgG control. mAb76 and mAb67 bound a known epitope of human SEMA4D, and competition with either would indicate functionality. Jurkat cells were preincubated on ice for 30 minutes with mAb76, mAb67, or mouse IgG control in FACS buffer (1 XPBS + 0.05% BSA +2mM EDTA) at 5ug/mL and 200,000 cells at 100 uL. This allows saturation binding of the mouse antibody to be achieved, thereby blocking binding to the epitope. Human test and control antibodies were preincubated with the secondary reagent goat anti-human Fc-Dylight 649 antibody (Jackson Immunoresearch 496-170) at 1ug/mL on ice for 30 min. After incubating Jurkat cells with mouse IgG antibodies, the cells were washed and incubated with the test antibody/secondary complex mixture on ice for 30 minutes. Cells were then washed twice with 200uL FACS buffer and resuspended in 250 uL PBS/1% BSA with PI and 0.5% paraformaldehyde fixing solution. After resuspension, cells were analyzed by flow cytometry on FACS CANTO II. PI negative populations were gated and Dylight649 transitions were recorded and evaluated. Percent binding inhibition has been calculated compared to human antibody binding to Jurkat cells preincubated with irrelevant mouse IgG.

MAb C2305 was selected for further characterization because it was able to cross-block previously characterized murine anti-SEMA 4D antibodies 67-2 and 76-1. See, for example, U.S. patent No. 8,496,938, the disclosure of which is incorporated by reference herein in its entirety.

The polynucleotide encoding MAb C2305 VH was cloned into a mammalian expression vector containing the human gamma-4 heavy chain constant region coding sequence, thereby creating a full-length heavy chain. The polynucleotide encoding MAb C2305 VL was cloned into a mammalian expression vector with human λ constant region coding sequence, creating a full length light chain. Expression vectors containing heavy and light chains were co-transfected into CHO-S cells. The monoclonal antibodies produced were secreted from the cells and collected after a 3-6 day expression period. The resulting MAb was purified using protein a chromatography and characterized. The resulting fully human MAb (MAb C2305) was demonstrated to be specific for SEMA4D by flow cytometry and by ELISA, and was shown to be able to compete with murine MAb67-2 for binding to SEMA 4D. The functional activity of MAb C2305 was further assessed in a receptor blocking assay according to the method in example 3 below. Receptor blockade was observed, but at a lower level compared to that of humanized MAb 2503 (U.S. patent No. 8,496,938, also known as VX15/2503) used as a comparator.

MAb C2305 was fully sequenced and then engineered for affinity improvement. Heavy chain complementarity determining region 3(HCDR3) was subjected to standard site-directed mutagenesis techniques and a novel fully human light chain variable region (VL) was identified from the light chain library according to the method described in U.S. patent application publication No. 2013-0288927-A1. Based on these changes, MAb with improved affinity, MAb D2517, was selected as the lead antibody. MAb D2517 was cloned, assembled, and expressed as fully human IgG4 antibody with lambda light chain. The VH, VL and CDR sequences of MAb D2517 are presented in table 2.

Table 2: MAb D2517 sequence

Example 2: binding characterization of MAb D2517

The binding characteristics of MAb D2517 were determined by BIACORE assay as follows. The antibody, both formulated in PBS, and the comparative antibody VX15/2503 were captured on the sensor at low density with a goat anti-human IgG Fc antibody, followed by flowing marmoset SEMA4D-His through the capture antibody at an antigen concentration range of 0-25 nm. The results are shown in table 3. The affinity of MAb D2517 for SEMA4D was similar to that of VX 15/2503.

Table 3: BIACORE assay results

MAb D2517 was tested for its ability to bind human SEMA4D, mouse SEMA4D, marmoset SEMA4D and cynomolgus SEMA4D by ELISA using the following method. Nunc maxisorp C well ELISA plates were coated overnight with 100. mu.L/well of CD100-His formulated in 1 XPBS. After overnight incubation, plates were washed and subsequently blocked with 200 uL/well PBS + 0.5% BSA +. 025% tween 20 for at least 1 hour at room temperature. After additional washing, 100 μ l/well dilution of MAb (100ng/mL) was added to each well of the ELISA plate, which was incubated for 2 hours at room temperature, followed by washing. Next, 100. mu.l/well of 1:10,000 goat anti-human Fc antibody (Jackson catalog number 109-035-098, lot 118460) was added to each well and the ELISA plates were incubated at room temperature for 1 hour. Plates were then washed and developed with 100 uL/well TMB substrate for 15 min. The detection reaction was terminated using 100 uL/well 2N sulfuric acid and the plate was read using a plate reader at a wavelength of 450-570 nM.

The results are shown in figure 1A (human SEMA4D), figure 1B (marmoset SEMA4D), figure 1C (cynomolgus SEMA4D) and figure 1D (mouse SEMA 4D).

Example 3: MAb D2517 blocks SEMA4D binding to its receptor

The ability of MAbD2517 to block SEMA4D from various species from binding to its receptor plexin B1 was tested as follows.

MAbD2517 and MAbVX15/2503 were formulated in acetate buffer. The ability of the antibody to block SEMA4D-His complex (human, cynomolgus monkey, mouse or rat SEMA4D) to bind 293PLXNB1 cells was compared in a triple dilution series. All antibodies were diluted from 1.5. mu.g/mL to 88ng/mL in 96-well plates and combined with 0.8ug/mL of the appropriate SEMA4D-His at 1:1 and incubated overnight at 4 ℃.

The following day, antibody/SEMA 4D-His complex or control was added to 2.5x10 in 96-well plates⁵293PLXNB1 cells and binding was allowed to occur at 4 ℃ for 30 min. After washing, cells were stained with anti-6 xHis antibody-APC (30 min, 4 ℃), washed, and analyzed by flow cytometry on a FACS Canto II. The results are shown in fig. 2A-2D. EC50 values were calculated from the curves and are shown in table 4.

Table 4: EC50 values (in nM) blocking SEMA4D binding to its receptor

SEMA4D-his	VX15/2503	MAb D2517
			Human SEMA4D	0.3	1.0
Cynomolgus monkey SEMA4D	0.5	1.2
			Mouse SEMA4D	0.4	1.2
Rat SEMA4D	0.6	1.5

Example 4: testing of murine chimeric equivalents of MAbD2517 in tumor models

The VH and VL amino acid sequences of MAb D2517 (SEQ ID NO:1 and SEQ ID NO:5) were inserted into a vector expressing murine IgG1 and lambda constant regions to generate chimeric antibody MAb D2585. The antibody was expressed in CHO-S cells and purified as described in example 1.

Balb/c female mice (n ═ 12) 6-8 weeks old were transplanted subcutaneously into the mammary fat pad of the mice with 30,000 tubo. a5 mammary tumor cells. Treatment with control mouse IgG1/2B8.1E7 or the anti-SEMA 4D chimeric antibody MAbD2585(10mg/kg, i.p., once a week for a total of 5 weeks) was initiated 7 days after inoculation. Tumors were measured with calipers, starting 11 days after implantation, 2 times/week. When the tumor volume reaches 800mm³Animals were sacrificed.

Tumor growth was measured by caliper gauge and the measurement was used to use the formula (w)²X l)/2, where w ═ the width of the tumor, i.e. the smaller measurement, and l ═ the length of the tumor, in mm. Mean tumor volume and defined as the tumor volume reached of 800mm³Kaplan Meier survival curves for the time of endpoint are shown in fig. 3A and fig. 3B, respectively. For mean tumor volume (p) used respectively<0.05) two-way analysis of variance (ANOVA) and for survival (p)<0.1) the average tumor volume and the survival rate are statistically significant.

The frequency of tumor regression in the Tubo tumor model was also measured and is shown in FIG. 3C. Regression is no palpable tumor, defined as tumor measured for at least two consecutive measurements<50mm³. Treatment with the chimeric antibody mab d2585 increased the number of regressions in mice carrying Tubo. As determined by Fisher's Exact test, as compared to control IgThe number of regressions in the group of mice treated with the chimeric antibody mab 2585 was statistically significant (p ═ 0.01).

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds semaphorin-4D (SEMA4D), said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, the HCDRs comprising amino acid sequences identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, or identical to SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, except for one, two, three or four amino acid substitutions in one, two or all three of the HCDRs; and wherein the VL comprises complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, the LCDRs comprising amino acid sequences identical to SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively, or identical to SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively, except for one, two, three, or four amino acid substitutions in one, two, or all three of the LCDRs.

2. The antibody or fragment thereof of claim 1, wherein the HCDR1, the HCDR2, and the HCDR3 comprise amino acid sequences identical to SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively, and wherein the LCDR1, the LCDR2, and the LCDR3 comprise amino acid sequences identical to SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively.

3. The antibody or fragment thereof of claim 1 or claim 2, wherein the VH further comprises the framework regions (HFW) HFW1, HFW2, HFW3, and HFW4, and wherein the VL further comprises the framework regions (LFW) LFW1, LFW2, LFW3, and LFW 4.

4. The antibody or fragment thereof of claim 3, wherein the framework region is derived from a human antibody.

5. The antibody or fragment thereof of claim 3, wherein the framework region is derived from a non-human antibody.

6. The antibody or fragment thereof of any one of claims 1 to 4, wherein the VH comprises the amino acid sequence SEQ ID NO 1.

7. The antibody or fragment thereof of any one of claims 1 to 4, wherein the VL comprises the amino acid sequence SEQ ID NO 5.

8. The antibody or fragment thereof of any one of claims 1 to 4, wherein said VH comprises the amino acid sequence SEQ ID NO 1 and said VL comprises the amino acid sequence SEQ ID NO 5.

9. An antibody or antigen-binding fragment thereof that specifically binds semaphorin-4D (SEMA4D), said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises an amino acid sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO 1.

10. The antibody or fragment thereof of claim 9, wherein the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 5.

11. An antibody or antigen-binding fragment thereof that specifically binds semaphorin-4D (SEMA4D), said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO 5.

12. The antibody or fragment thereof of claim 11, wherein the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID No. 5.

13. An antibody or antigen-binding fragment thereof that specifically binds semaphorin-4D (SEMA4D), said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO 1; and wherein the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO 5.

14. The antibody or fragment thereof of claim 13, wherein said VH comprises the amino acid sequence of SEQ ID No. 1, and wherein said VL comprises the amino acid sequence of SEQ ID No. 5.

15. The antibody or fragment thereof of any one of claims 1 to 14, further comprising a heavy chain constant region or fragment thereof fused to the C-terminus of said VH.

16. The antibody or fragment thereof of claim 15, wherein the heavy chain constant region or fragment thereof is a human heavy chain constant region.

17. The antibody or fragment thereof of claim 16, wherein the heavy chain constant region or fragment thereof is a human IgG4 constant region.

18. The antibody or fragment thereof of claim 15, wherein the heavy chain constant region or fragment thereof is a non-human heavy chain constant region.

19. The antibody or fragment thereof of claim 18, wherein the heavy chain constant region or fragment thereof is a murine IgG1 constant region.

20. The antibody or fragment thereof of any one of claims 1 to 19, further comprising a light chain constant region or fragment thereof fused to the C-terminus of the VL.

21. The antibody or fragment thereof of claim 20, wherein the light chain constant region or fragment thereof is a human light chain constant region.

22. The antibody or fragment thereof of claim 21, wherein the light chain constant region or fragment thereof is a human λ light chain constant region.

23. The antibody or fragment thereof of claim 20, wherein the light chain constant region or fragment thereof is a non-human light chain constant region.

24. The antibody or fragment thereof of claim 23, wherein the light chain constant region or fragment thereof is a murine lambda light chain constant region.

25. The antibody or fragment thereof of any one of claims 1 to 24, which is a Fab fragment, Fab 'fragment, F (ab')₂A fragment, Fd fragment, single chain Fv fragment (scFv), or disulfide linked Fv fragment (sdFv).

26. The antibody or fragment thereof of any one of claims 1 to 25, which is multispecific.

27. The antibody of any one of claims 1 to 26, which specifically binds to human SEMA4D, mouse SEMA4D, rat SEMA4D, or non-human primate SEMA 4D.

28. The antibody or fragment thereof of claim 27, wherein the non-human primate SEMA4D is cynomolgus SEMA4D or marmoset SEMA 4D.

29. The antibody or fragment thereof of claim 27 or claim 28, which specifically binds SEMA4D with an affinity characterized by a dissociation constant KD of no greater than 500nM, 100nM, 50.0nM, 40.0nM, 30.0nM, 20.0nM, 10.0nM, 9.0nM, 8.0nM, 7.0nM, 6.0nM, 5.0nM, 4.0nM, 3.0nM, 2.0nM, 1.0nM, 0.50nM, 0.10nM, 0.050nM, 0.01nM, 0.005nM, or 0.001 nM; and wherein said SEMA4D is human SEMA4D, mouse SEMA4D, cynomolgus monkey SEMA4D, marmoset SEMA4D, or any combination thereof.

30. The antibody or fragment thereof of any one of claims 1 to 29, which inhibits the binding of SEMA4D to the SEMA4D receptor.

31. The antibody or fragment thereof of claim 30, wherein the SEMA4D receptor is plexin-B1, plexin-B2, CD72, or a combination thereof.

32. The antibody or fragment thereof of any one of claims 1 to 31, which elicits minimal or no anti-antibody immune response upon administration to a human subject.

33. The antibody or fragment thereof of any one of claims 1 to 32, further comprising a heterologous moiety fused or conjugated thereto.

34. The antibody or fragment thereof of claim 33, wherein the heterologous moiety comprises a polypeptide, a cytotoxic agent, a therapeutic agent, a prodrug, a lipid, a carbohydrate, a nucleic acid, a detectable label, a polymer, or any combination thereof.

35. The antibody or fragment thereof of claim 34, wherein the polypeptide comprises a binding molecule, an enzyme, a cytokine, a lymphokine, a hormone peptide, or any combination thereof.

36. The antibody or fragment thereof of claim 34, wherein the cytotoxic agent comprises a radionuclide, a biotoxin, an enzymatically active toxin, or any combination thereof.

37. The antibody or fragment thereof of claim 34, wherein the detectable label comprises an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or any combination thereof.

38. A composition comprising the antibody or fragment thereof of any one of claims 1 to 37, and a carrier.

39. An isolated polynucleotide or combination of polynucleotides comprising one or more nucleic acid sequences encoding the antibody or fragment thereof or subunit thereof of any one of claims 1 to 37.

40. The polynucleotide or combination of polynucleotides of claim 39, comprising a nucleic acid sequence encoding the VH of the antibody or fragment thereof.

41. The polynucleotide or combination of polynucleotides of claim 39, comprising a nucleic acid sequence encoding the VL of the antibody or fragment thereof.

42. The polynucleotide or combination of polynucleotides of claim 39, comprising a nucleic acid sequence encoding the VH of the antibody or fragment thereof and a nucleic acid sequence encoding the VL of the antibody or fragment thereof.

43. The polynucleotide or combination of polynucleotides of claim 42, wherein said VH-encoding nucleic acid sequence and said VL-encoding nucleic acid sequence are located on the same vector.

44. The vector of claim 43.

45. The polynucleotide or combination of polynucleotides of claim 42, wherein said VH-encoding nucleic acid sequence and said VL-encoding nucleic acid sequence are located on separate vectors.

46. The vector of claim 45.

47. The vector of claim 44 or claim 46, further comprising a genetic element to allow expression of the antibody or fragment thereof.

48. A host cell comprising the polynucleotide or combination of polynucleotides of any one of claims 39 to 43 or 45, or the vector of claim 44 or claim 46.

49. A method of producing the antibody or fragment thereof of any one of claims 1 to 37, comprising culturing the host cell of claim 48, and recovering the antibody or fragment thereof.

50. A method of neutralizing SEMA4D in a human subject comprising administering to the human subject the antibody or fragment thereof of any one of claims 1 to 37 or the composition of claim 38.

51. The method of claim 50, wherein the human subject is in need of treatment for an autoimmune disease or disorder, an inflammatory disease or disorder, cancer, a neuroinflammatory disease or disorder, a neurodegenerative disease or disorder, or any combination thereof.

52. The method of claim 51, wherein the neuroinflammatory disease or disorder is multiple sclerosis.

53. The method of claim 51, wherein the neurodegenerative disease or disorder is stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Down's syndrome, ataxia, Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), HIV-associated cognitive impairment, CNS lupus, mild cognitive impairment, or a combination thereof.

54. The method of claim 51, wherein the autoimmune disease or the inflammatory disease is arthritis.

55. The method of claim 54, wherein the autoimmune disease or the inflammatory disease is rheumatoid arthritis.